Hindawi Publishing CorporationScientificaVolume 2013 Article ID 635203 22 pageshttpdxdoiorg1011552013635203

Review ArticleMelanoma From Melanocyte to Genetic Alterations andClinical Options

Corine Bertolotto12

1 INSERM U1065 (Equipe 1) C3M 06204 Nice France2 University of Nice Sophia-Antipolis UFR Medecine 06204 Nice France

Correspondence should be addressed to Corine Bertolotto bertolotunicefr

Received 21 October 2013 Accepted 7 November 2013

Academic Editors M Alaibac P Auberger H Sowter and R Teasdale

Copyright copy 2013 Corine Bertolotto This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Metastatic melanoma remained for decades without any effective treatment and was thus considered as a paradigm of cancerresistance Recent progress with understanding of the molecular mechanisms underlying melanoma initiation and progressionrevealed that melanomas are genetically and phenotypically heterogeneous tumors This recent progress has allowed for thedevelopment of treatment able to improve for the first time the overall disease-free survival of metastatic melanoma patientsHowever clinical responses are still either too transient or limited to restricted patient subsets The complete cure of metastaticmelanoma therefore remains a challenge in the clinic This review aims to present the recent knowledge and discoveries of themolecular mechanisms involved in melanoma pathogenesis and their exploitation into clinic that have recently facilitated bench tobedside advances

1 The Melanocytes From Photoprotectionto Cancer

11 Melanocyte Development Melanoblasts undifferentiatedand unpigmented precursors migrate from the neural crestto their final destination the epidermis and hair follicleswhere they differentiate and becomematuremelanocytes ableto synthesize and transfer melanin pigment to neighbouringkeratinocytes (Figure 1) Melanocytes are also found in thestria vascularis of the inner ear cochlea where they areinvolved in the production of endolymph along with ionexchange essential for hearing Melanocytes are also locatedin the iris and in the choroid where pigments are involved inthe formation behind the retina of the darkroom which isnecessary for the vision This review will focus on cutaneousmelanocytes only

During embryogenesis the survival and migration ofmelanocytes rely on signaling pathways such as Wing-less signaling (Wnt)120573-catenin the endothelin B receptorand its ligand endothelin-3 the receptor tyrosine kinaseKIT and its ligand KIT-ligandSCF (stem cell factor)NOTCH [1 2] and transcription factors activity such as

paired box gene 3 (PAX3) SRY (sex-determining regionY)-box10 (SOX10) hairyenhancer of split (HES1) andmicrophthalmia-associated transcription factor (MITF) [3ndash7] In humans mutations in the genes encoding KIT PAX3SOX10 and MITF are the cause of various diseases suchas Piebaldism Waardenburg or Tietz syndromes which arecharacterized by patchy depigmentation Patients sufferingfrom the Waardenburg or Tietz syndromes are also charac-terized by profound deafness [8 9]

A pool of melanocytes with stemness properties (MSC)remains in the lower permanent portion (bulge) of the hairfollicle where these MSC can segregate during the haircycle into mature melanocytes that color the new growinghair and into melanocyte-stem cells to maintain the poolof undifferentiated melanocytes Maintenance of MSC alsodepends on the above signaling pathway and transcriptionfactors [1] Mouse models revealed that MSC are char-acterized by a low-level expression of Mitf cKit Sox10Lef1 and Ednrb which control melanocyte proliferation anobservation consistent with the quiescence status of theMSCOn the other hand Pax3 and Dct expression were foundupregulated [10] Although the mechanisms which control

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EpidermisMelanocyte

Keratinocytes

DermisFibroblasts

Dorso-ventral migration

PAX3 SOX10 ED-3Endrb C-KIT and MITF

Melanosome

Tyrosine DOPA DHICATyrosinase DCT TYRP1

Melanin

Neural crest

Melanoblasts

DOPAChrome

Figure 1 General overview of melanocyte physiology Melanocytes derived from the neural crest in the form of undifferentiated andunpigmented precursors the melanoblasts migrate to their final destination the epidermis where they synthesize melanin in melanosomesPax3 Sox10 endothelin3 (ED-3) and its receptor (Endrb) c-Kit and Mitf play a critical role in the development of melanocytes Melanin isthen transferred to neighboring keratinocytes to ensure skin protection against the deleterious effect of ultraviolet radiation

this program remain to be fully elucidated an explanationcould be elevated expression ofWnt signaling inhibitors suchas Dkk3 Sfrp1 and Dab2 in the microenvironment [1 11]

12 Physiological Role of Melanocytes The main physiolog-ical function of skin melanocytes is to produce melaninpigments The pigments are synthesized within specializedorganelles calledmelanosomes through an enzymatic cascadeinvolving tyrosinase tyrosinase-related protein-1 (TYRP1)and tyrosinase-related protein 2dopachrome tautomerase(DCT) Two types of pigments are produced the brownblackpigment eumelanin which displays photoprotective featuresand the orangeyellow pigment pheomelanin endowed withpoor photoprotective properties (Figure 1) Pigmentation isa heritable trait being regulated by genetic factors but theamount type and distribution of melanins in the skinhair and eyes can also be influenced by environmental andendocrine factors Pigments have an extremely importantrole in our organism because they provide an efficient protec-tion against the harmful effect of ultraviolet radiation (UVR)By absorbing and scattering UVRmelanin reduces the UVR-induced cellular DNA damage and genomic instability Thisprocess is evenmore important upon tanning purposes and itresponds to damage fromUVR by producingmore pigmentsThe central transducer of this response is p53 activation inkeratinocytes which uponUVR stimulates the transcriptionof proopiomelanocortin (POMC) the precursor of hormones

such as 120572-melanocyte stimulating hormone (120572MSH) oradrenocorticotropic hormone (ACTH) endowed with pro-pigmenting activities [12] 120572MSH through activation of itsmelanocortin-1 receptor (MC1R) activates the cAMPproteinkinase A (PKA)CREB signaling pathway and enhances thelevel of the transcription factor MITF [13 14] MITF iscritically required for pigment production and a reductionin its activity is coupled to reduce melanin synthesis andpigmentation [15] MITF also regulates expression of genesinvolved in melanosome biogenesis [16 17] melanin synthe-sis [18 19] and melanosome trafficking [20 21] ThereforeMITF coordinates an integrated cellular response for pigmentproduction and function

In addition to increased pigmentation an increased num-ber of melanocytes is found in UVR-exposed skin [22ndash24]a process that likely contributes to enhance photoprotectionof the skin through increased ability to provide skin withpigment The proliferative ability of melanocytes mainlydepends on keratinocyte-released factors such as 120572MSHendothelin granulocyte-macrophage colony-stimulating fac-tor (GM-CSF) steel factor leukemia inhibitory factor (LIF)basic fibroblast growth factor (bFGF) and hepatocyte growthfactor (HGF) [25] which secretion may be stimulated byUVR The growth factors hormones and ligands bind andactivate their respective receptors that are connected to theMAPKERK and PI3KAKT signaling cascades Our labprovided the first demonstration of BRAF expression and itsimplication in melanocyte function by showing that BRAF

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activation via the 120572MSHcAMP cascade led to an increase ofproliferation upon the MEKERK signaling pathway activa-tion [26] This report was the beginning of what is now thebiggest focus in the melanocyte field the role of BRAF inmelanoma disease Activation of the ERK and PI3K signalingpathways are also associated with a survival program Indeedconsidering the key role of melanocytes in protecting ourbody against the noxious effect of UVR melanocytes havedeveloped potent antiapoptoticmechanisms involved in theirresistance to the UVR-induced DNA damage and cell deathIn this context MITF whose expression is increased uponUVR controls the expression of antiapoptotic genes such asBCL2 [27] BCL2A1 [28] andML-IAP [29] and several genesinvolved in DNA repair [30]

Exposure of human skinto sunlight has positive healtheffects in its ability to boost the bodyrsquos vitamin D supplywhich is essential for overall body health The avoidanceof all direct sun exposure increases the risk of vitamin Ddeficiency which can have serious consequences amongwhich increased risks of deadly cancers [31 32] Howeverfrequent and intense exposure to UV radiation sunlightespecially in childhood is the major environmental riskfactor for melanoma development

These observations illustrate how important is the del-icate balance between differentiation and proliferationsur-vival of melanocytes

13 Melanoma Disease Cutaneous metastatic melanoma(CMM) deriving from melanocyte transformation in 75 ofthe cases and from preexisting nevi in 25 of the cases isthe most deadly form of skin cancer Melanoma accounts forless than 5 of skin cancers but is responsible for 80 ofskin cancer related deathsMelanoma incidence in Caucasianpopulation has increased dramatically worldwide during thepast several decades Melanoma is a cancer with a relativelygood prognosis when diagnosed early at a cutaneous local-ized stage Patients with stage 0I thin lesions can usuallybe cured with surgical excision Their 5-year survival rateranges from 90 to 100 However the prognosis worsensthe deeper the lesion extends beneath the skin becauseof melanomarsquos propensity to invade and to metastasizeIndividuals with thick melanomas have an increased riskto develop lymph node and visceral metastases Metastaticmelanoma cannot be completely removed by surgery andmetastatic cells display extreme resistance to all types oftreatment Patientswithmetastaticmelanomas have amediansurvival rate that typically ranges from six to ten months

Melanomagenesis is a complex phenomenon in whichenvironmental genetic and host factors play a role Datafrom the clinic epidemiology andmore recently from geneticreveal that melanomas are heterogeneous tumors harboringvarious genetic alterations developing at different body sitesand on sun-exposed and non sun-exposed regions suggest-ing that melanoma arises from divergent causal pathwaysCurtin et al proposed a molecular classification based onthe sites where the melanoma occurs the genetic alterationsand the sun exposure history [33] Mutations in BRAF weresignificantly more common in melanomas located in areas

without chronic sun-induced damage Melanomas arising inchronically sun-damaged skin mucosal surfaces and acralskin were characterized by wild-type BRAF and wild-typeNRAS but exhibited alterations in KIT

131 The Constitutional Risk Factors We are not all equal infront of the sun Major risk factors include skin phototypeand skin reaction to sun exposure according to the phototypea high number of nevidysplasic nevi and a personal andfamilial history of melanoma

Subjects with redblond hair blue eyes fair skin anddeveloping sunburns have higher melanoma risk than sub-jects with brown haireyes and skin that tan easily Theformer produce little or no eumelanin and are thereforemuch less protected from the noxious effects of UV radiationSubjects with gt50 nevi or having at least five atypical molesexhibit fivefold higher risk of developing melanoma Thepresence of large congenital nevi is also a melanoma riskfactor [34] Finally approximately 10ofmelanomas occur ina familial context defined by at least two melanomas withintwo or more members of the same family Rare deleteriousgerminal mutations in the cell cycle regulators CDKN2Aand cyclin dependent-kinase 4 (CDK4) have been shown toconfer a high cutaneous malignant melanoma risk [35 36]Additionally the frequent allelic germinal variants (MC1RASIP MTAP MATP and Casp8) have been identified aslow-risk susceptibility genes or as modifiers of high-riskgenes [37 38] Recently a germinal mutation in the mastergene of melanocyte homeostasis microphthalmia-associatedtranscription factor (MITF) has been identified and shownto increase the risk of developing melanoma [39ndash42] Finallysubjects who have had a melanoma have an approximately9-fold increased risk of developing subsequent melanomacompared with the general population [43]

132 Acquired Risk Factors Epidemiologic studies revealedthat sun exposure is the major known environmental factorassociated with development of melanoma According to theworld health organization (WHO) changes in sun exposurehabits and attitudes that often resulted in excessive UVexposure were recorded in the recent decades and is themaincause of the increase in skin cancer number

Excessive sunlight exposure associated with sunburnespecially in childhood and early adolescence years starts thetransformation of benign melanocytes into a malignant phe-notype Results fromNoonan et al who used a mouse modelexpressing HGF derived by the metallothionein promoterconstitute proof-of-concept [44] A single UV dose deliveredat the neonatal stage was sufficient to induce melanoma inHGFSF-transgenic mice after a relatively short latent periodand with high cumulative incidence Noteworthy the doseroughly administrated to mice in this study corresponds toa sunburning dose of natural sunlight at midlatitudes inmidsummer No melanoma was detected when UVR wasdelivered at the adult stage

Furthermore one personrsquos risk of developing melanomadoubles if they have more than five sunburns at any age Themajority of melanomas are located on intermittently exposed

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body sites such as the trunk inmen and lower limbs inwomen[45]

Sunrsquos ultraviolet rays are divided into wavelength rangesidentified as UVA (315 to 400 nm) UVB (280 to 315 nm) andUVC (100 to 280 nm) UVC is filtered by the stratosphericozone layer and in theory it does not reach earthrsquos surfaceOfthe UV solar radiation that does reach the earth UVA (95)and UVB (5) promote deleterious effects on proteins andnucleic acids UVB is thought to be more carcinogenic thanUVA by inducing the formation of cyclobutane pyrimidinedimers (CPD) and 6-pyrimidine 4-pyrimidone photoprod-ucts [46] while UVA mainly produces oxidative stress [47]The frequency of CPD generation is three times higher andthey are less efficiently repaired than 6-4 photoproducts TheUVB-induced lesions generate typical genetic mutations Cto T and CC to TT transitions called the ldquoUVB signaturemutationsrdquo Both UVA and UVB can also trigger DNAdamage through oxidative stress and indirectly damage DNAand cause genetic alterations Experiments using animalmodels showed that UVB but not UVA was melanomagenic[22 48]

Therefore UVR may contribute to melanoma develop-ment through combined genotoxic and mitogenic effectsin melanocytes DNA repair-deficient xeroderma pigmento-sum A (Xpa) mice display higher melanocyte proliferationinduction by UVB exposure [23] and individuals with XPmutations display a greatly elevated incidence of skin cancersincluding melanoma [49] thereby indicating that DNA-repair genes play an active role in melanoma initiation DNArepair genes are also involved in melanoma progression byconferring a sort of metastatic genome stabilization duringthe metastatic process [50] The molecular mechanism bywhich these genes are controlled are poorly elucidated yetfunctional genomic experiments unveiled that the mastergene ofmelanocyte homeostasisMITF controls the transcrip-tion of several genes involved in DNA replication and repair[30]

Together these observations point to the importanceof effective DNA damage repair and genomic stability inmelanocyte transformation and melanoma initiation

The absence of UV ldquosignaturerdquo mutations in genes rele-vant to melanoma such as BRAF NRAS CDKN2A or evenp53 which harbor typical UV signature in nonmelanomaskin cancers [51] questioned for years the role of UVR inmelanoma disease Numerous recent publications solved thismystery using ldquodeep sequencing approachesrdquo by showingfrequent UV signature mutation in the genome of differentmelanoma types thereby providing the first genomic evi-dence for a direct mutagenic role of UV light in melanomapathogenesis

In a first study the authors sequenced the coding exonsof 518 kinases in several cancers including melanoma andgenome sequencing of a malignant melanoma and a lym-phoblastoid cell line from the same person revealed thatthe dominant mutational signature was CgtT transversionreflecting DNA damage due to ultraviolet light exposure[52 53] Of the 33345 somatic base substitutions reportedby Pleasance et al almost 25000 were CgtT changes and360 were CCgtTT changes Additionally a high frequency

of GgtT substitutions that might reflect transversion ensuingoxidative DNA damage has been detected [54] A highfrequency of CgtT transversions was nevertheless also iden-tified in non-sun-related cancers such as glioma gastric orcolorectal cancers More evidence came from three otherstudies in which they compared the mutational status ofmelanomas from different body areas such as melanomasfrom the trunk versus sun-shielded acral mucosal and uvealmelanomas [55ndash57] The rate of point mutations was thelowest in primaries from non-ultraviolet-exposed hairlessskin of the extremities (3 and 14 per megabase (Mb) ofgenome) The mutational rate was scored intermediate inthose originating from hair-bearing skin of the trunk (5ndash55 per Mb) and highest in a patient with chronic sunexposure (111 per Mb) [55] Moreover these studies pointedout to mutations affecting the biological function of proteinssuch as PREX2 (phosphatidylinositol-345-trisphosphate-dependent Rac exchange factor 2)-a PTEN-interacting pro-tein and negative regulator of PTEN which is mutated in14 [55] PPP6C encoding a serinethreonine phosphatasemutated in 9ndash124 and RAC1 encoding a GTPase of theRAS superfamily of small GTP-binding proteins mutated in5ndash92 [56 57] of sun-exposedmelanoma cases NoteworthyPPP6C mutations were more frequent in melanomas thatwere mutated for both BRAF and NRAS while RAC1 muta-tions were more frequent in melanomas that were wild-typefor both BRAF and NRAS [56 57] Therefore these studiesfirmly implicatedUV irradiation inmelanomas and providedpotential new therapeutic options

2 Melanoma Disease From Molecular Biologyto Clinic

Tumor growth is the result of genetic andor epigeneticalterations in key genes (ldquodriverrdquo genes) controlling processessuch as proliferation apoptosis senescence and responseto DNA damage These changes lead to the synthesis ofmodifiedactivated (for oncogenes) or hypofunctionalabsent(for tumor suppressor genes or DNA repair genes) proteinsChanges in the stoichiometry and normal biological behaviorof these proteins within the cells will promote an accelerationof the tumor progression At the initiation stage the geneticalteration can be germline Aswe know that the prognosis of apatient is closely linked to its early diagnosis identification ofcancer predisposing genes is crucial to identify and monitorat-risk patients This chapter provides an overview of the keymolecular proteins and associated pathways implicated in theacquisition of the malignant melanoma phenotype

21 Melanoma Susceptibility Genes Historically high-riskgermline mutations in cancer predisposing genes (oncogenesor tumor suppressor genes) were discovered through linkageanalysis in large pedigrees showing Mendelian-like modeof inheritance During the last five years genome-wideassociation studies (GWAS) have identified common SNPsassociated with a low risk of developing cancers includingmelanoma [58] However formany cancers as for other com-plex diseases and human traits the known loci explain only

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relatively small fractions of the total genetic variance andthe identification of genetic variants with low allele frequencyconferring a moderate-risk of cancer through these classicalapproaches is much more challenging Therefore mutationscreening of candidate genes (or of the entire exome nowachievable thanks to the latest improvement in sequencingtechnologies) in carefully selected at-risk patients coupledwith appropriate tools to assess the pathogenicity of thegenetic variants (in silico and in vitro assays) is an importantcomplementary approach of choice to identify the missingheritability

Recent studies have demonstrated that common diseasescan be due to dysfunctional variants with a wide spectrumof allele frequencies ranging from rare to common Thisis the case for example in breast cancer where high-riskmutations inBRCA1BRCA2 PTEN andTP53 intermediate-risk variants in genes of the DNA repair pathways (ATMCHEK2 BRIP1 and PALB2) and low-risk SNPs (in FGFR2TOX3 CASP8MAP3K1 and LSP1) have been identified [59]

Until very recently these rare sometimes privatemoderate-risk susceptibility alleles have been identifiedthrough resequencing of candidate genes selected on thebasis of biological plausibility A candidate gene approachhas been applied to identify the MITF E318K mutationIn this particular case the fact that two studies reportedthe same recurrent MITF mutation in independent patientseriespopulations using an agnostic genome-wide approach(massive parallel exome sequencing approach plus genotyp-ing in larger melanoma series familial CMM where multipleprimaries often occur) reinforces the notion of the existenceof variants with low minor allele frequency that could havesubstantial effects (Figure 2)

211 Genes with High Penetrance Rare alleles of CDKN2Aand CDK4 genes have been identified in familial formsof melanoma among patients who have had melanomaMutations or deletions in these genes confer an elevatedrisk of developing melanoma These genes are involved incell cycle arrest and cellular senescence Their importance isrevealed by the fact that germline and somatic mutations inCDKN2A and CDK4 directly or in their associated signalingpathways are almost invariably found in melanomas

The CDKN2A Locus CDKN2A (Cyclin-Dependent KinaseInhibitor 2A) is located at the 9p21 locus and encodes twotumor suppressor proteins p16INK4a (Inhibitor of Kinase a)and p14ARF (Alternative Reading Frame) p16INK4A is tran-scribed from exons 1120572 2 and 3 with exon 3 encoding onlyfour amino acids p14ARF is encoded by an alternative exon 1(1120573) spliced to CDKN2A exon 2 in an alternate reading frame(ARF) The p16INK4a and p14ARF transcripts are translatedin different reading frames thus the two proteins have nohomology at the amino acid level [60] In familial melanomawhich represents 8 to 12 of all melanomas [61] germlinemutations in the CDKN2A gene are found in 20ndash40of cases[62] These mutations can affect p16INK4a p14ARF or bothproteins p16INK4a interacts specifically with both CDK4 andCDK6 and blocks their association with D-type complexes

[63] Thus the loss of function of p16INK4a promotes CDK4and CDK6 activation resulting in hyperphosphorylation ofpRB and the activation of the transcription factor E2F1E2F1 mediates the transcription of S phase promoting genesthereby promoting cell proliferation

Evidence of a role for p16INK4a in human melanomaincludes frequent somatic genetic and epigenetic alterationsin human melanoma samples Indeed p16INK4a gene is lostin 50 of melanoma cases inactivated by point mutations inapproximately 9 of tumors or inactivated by methylationof its promoter in about 10 of melanoma cases [64]p16INK4a loss of function promotes senescence bypass andmelanocyte immortalization [65 66] Senescence is a pro-gram that represents a potent barrier against tumorigenesisby preventing the proliferation of cells at risk for neoplasictransformation The reintroduction of a functional copy ofp16INK4a in melanoma cell lines induced a significant changein morphology associated with dendricity a parameter ofdifferentiation and decreased cell growth [67]

The collaboration of oncogenic NRASQ61K and p16INK4a

loss of expression is sufficient to cause melanoma formationin mice supporting the critical role of p16INK4a in melanomaaetiology [68]

Although p16INK4a was thought to be the predominanttumor suppressor at 9p21 observations from melanoma-prone families in which exon 1120573 germline deletion or muta-tion in either the coding region or splice donor site of thisexon have been reported supporting a p16INK4a-independenttumor suppressor role for p14ARF [69ndash71] A comprehensiveanalysis of the pattern of genetic and epigenetic alterations tothe p16INK4a and p14ARF tumor suppressor loci in melanomarevealed that p14ARF is frequently inactivated [72] Sup-porting the role of p14ARF in melanomagenesis murinemelanoma models demonstrated that specific inactivationof p14ARF (p19ARF in mouse) enhanced melanoma devel-opment [73 74] Although p14ARF is mainly known tofunction by preventing p53 degradation by the E3 ubiquitin-protein ligase MDM2 (Mouse Double Minute 2) in mousecontext melanomagenesis in ARF-depleted background isp53-independent [73 74]

Consistently activating mutations of BRAF and loss offunctional p16INK4a and p14ARF were detected in themajorityof melanomas [75]

CDK4 Germline mutations in the gene encoding cyclindependent kinase 4 (CDK4A) have been identified in avery small percentage of familial melanoma [36 76] Themutation of arginine at position 24 into cysteine (CDK4R24C)or histidine (CDK4R24C) renders the protein insensitive toregulation by p16INK4a but preserves interaction betweenCDK4 and cyclin D1 leading to constitutive activation of thecomplex and aberrant proliferation through retinoblastomaprotein inactivation and E2F activation CDK4R24C facilitatestumorigenesis of melanocytes transplanted into nude miceand causes escape from cellular senescence [77] Mousemodels show that the CDK4R24C form enhances melanomapenetrance in cooperationwith the oncogenicHRasG12V [78]

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Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

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ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

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pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

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p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

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increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

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2 Scientifica

EpidermisMelanocyte

Keratinocytes

DermisFibroblasts

Dorso-ventral migration

PAX3 SOX10 ED-3Endrb C-KIT and MITF

Melanosome

Tyrosine DOPA DHICATyrosinase DCT TYRP1

Melanin

Neural crest

Melanoblasts

DOPAChrome

Figure 1 General overview of melanocyte physiology Melanocytes derived from the neural crest in the form of undifferentiated andunpigmented precursors the melanoblasts migrate to their final destination the epidermis where they synthesize melanin in melanosomesPax3 Sox10 endothelin3 (ED-3) and its receptor (Endrb) c-Kit and Mitf play a critical role in the development of melanocytes Melanin isthen transferred to neighboring keratinocytes to ensure skin protection against the deleterious effect of ultraviolet radiation

this program remain to be fully elucidated an explanationcould be elevated expression ofWnt signaling inhibitors suchas Dkk3 Sfrp1 and Dab2 in the microenvironment [1 11]

12 Physiological Role of Melanocytes The main physiolog-ical function of skin melanocytes is to produce melaninpigments The pigments are synthesized within specializedorganelles calledmelanosomes through an enzymatic cascadeinvolving tyrosinase tyrosinase-related protein-1 (TYRP1)and tyrosinase-related protein 2dopachrome tautomerase(DCT) Two types of pigments are produced the brownblackpigment eumelanin which displays photoprotective featuresand the orangeyellow pigment pheomelanin endowed withpoor photoprotective properties (Figure 1) Pigmentation isa heritable trait being regulated by genetic factors but theamount type and distribution of melanins in the skinhair and eyes can also be influenced by environmental andendocrine factors Pigments have an extremely importantrole in our organism because they provide an efficient protec-tion against the harmful effect of ultraviolet radiation (UVR)By absorbing and scattering UVRmelanin reduces the UVR-induced cellular DNA damage and genomic instability Thisprocess is evenmore important upon tanning purposes and itresponds to damage fromUVR by producingmore pigmentsThe central transducer of this response is p53 activation inkeratinocytes which uponUVR stimulates the transcriptionof proopiomelanocortin (POMC) the precursor of hormones

such as 120572-melanocyte stimulating hormone (120572MSH) oradrenocorticotropic hormone (ACTH) endowed with pro-pigmenting activities [12] 120572MSH through activation of itsmelanocortin-1 receptor (MC1R) activates the cAMPproteinkinase A (PKA)CREB signaling pathway and enhances thelevel of the transcription factor MITF [13 14] MITF iscritically required for pigment production and a reductionin its activity is coupled to reduce melanin synthesis andpigmentation [15] MITF also regulates expression of genesinvolved in melanosome biogenesis [16 17] melanin synthe-sis [18 19] and melanosome trafficking [20 21] ThereforeMITF coordinates an integrated cellular response for pigmentproduction and function

In addition to increased pigmentation an increased num-ber of melanocytes is found in UVR-exposed skin [22ndash24]a process that likely contributes to enhance photoprotectionof the skin through increased ability to provide skin withpigment The proliferative ability of melanocytes mainlydepends on keratinocyte-released factors such as 120572MSHendothelin granulocyte-macrophage colony-stimulating fac-tor (GM-CSF) steel factor leukemia inhibitory factor (LIF)basic fibroblast growth factor (bFGF) and hepatocyte growthfactor (HGF) [25] which secretion may be stimulated byUVR The growth factors hormones and ligands bind andactivate their respective receptors that are connected to theMAPKERK and PI3KAKT signaling cascades Our labprovided the first demonstration of BRAF expression and itsimplication in melanocyte function by showing that BRAF

Scientifica 3

activation via the 120572MSHcAMP cascade led to an increase ofproliferation upon the MEKERK signaling pathway activa-tion [26] This report was the beginning of what is now thebiggest focus in the melanocyte field the role of BRAF inmelanoma disease Activation of the ERK and PI3K signalingpathways are also associated with a survival program Indeedconsidering the key role of melanocytes in protecting ourbody against the noxious effect of UVR melanocytes havedeveloped potent antiapoptoticmechanisms involved in theirresistance to the UVR-induced DNA damage and cell deathIn this context MITF whose expression is increased uponUVR controls the expression of antiapoptotic genes such asBCL2 [27] BCL2A1 [28] andML-IAP [29] and several genesinvolved in DNA repair [30]

Exposure of human skinto sunlight has positive healtheffects in its ability to boost the bodyrsquos vitamin D supplywhich is essential for overall body health The avoidanceof all direct sun exposure increases the risk of vitamin Ddeficiency which can have serious consequences amongwhich increased risks of deadly cancers [31 32] Howeverfrequent and intense exposure to UV radiation sunlightespecially in childhood is the major environmental riskfactor for melanoma development

These observations illustrate how important is the del-icate balance between differentiation and proliferationsur-vival of melanocytes

13 Melanoma Disease Cutaneous metastatic melanoma(CMM) deriving from melanocyte transformation in 75 ofthe cases and from preexisting nevi in 25 of the cases isthe most deadly form of skin cancer Melanoma accounts forless than 5 of skin cancers but is responsible for 80 ofskin cancer related deathsMelanoma incidence in Caucasianpopulation has increased dramatically worldwide during thepast several decades Melanoma is a cancer with a relativelygood prognosis when diagnosed early at a cutaneous local-ized stage Patients with stage 0I thin lesions can usuallybe cured with surgical excision Their 5-year survival rateranges from 90 to 100 However the prognosis worsensthe deeper the lesion extends beneath the skin becauseof melanomarsquos propensity to invade and to metastasizeIndividuals with thick melanomas have an increased riskto develop lymph node and visceral metastases Metastaticmelanoma cannot be completely removed by surgery andmetastatic cells display extreme resistance to all types oftreatment Patientswithmetastaticmelanomas have amediansurvival rate that typically ranges from six to ten months

Melanomagenesis is a complex phenomenon in whichenvironmental genetic and host factors play a role Datafrom the clinic epidemiology andmore recently from geneticreveal that melanomas are heterogeneous tumors harboringvarious genetic alterations developing at different body sitesand on sun-exposed and non sun-exposed regions suggest-ing that melanoma arises from divergent causal pathwaysCurtin et al proposed a molecular classification based onthe sites where the melanoma occurs the genetic alterationsand the sun exposure history [33] Mutations in BRAF weresignificantly more common in melanomas located in areas

without chronic sun-induced damage Melanomas arising inchronically sun-damaged skin mucosal surfaces and acralskin were characterized by wild-type BRAF and wild-typeNRAS but exhibited alterations in KIT

131 The Constitutional Risk Factors We are not all equal infront of the sun Major risk factors include skin phototypeand skin reaction to sun exposure according to the phototypea high number of nevidysplasic nevi and a personal andfamilial history of melanoma

Subjects with redblond hair blue eyes fair skin anddeveloping sunburns have higher melanoma risk than sub-jects with brown haireyes and skin that tan easily Theformer produce little or no eumelanin and are thereforemuch less protected from the noxious effects of UV radiationSubjects with gt50 nevi or having at least five atypical molesexhibit fivefold higher risk of developing melanoma Thepresence of large congenital nevi is also a melanoma riskfactor [34] Finally approximately 10ofmelanomas occur ina familial context defined by at least two melanomas withintwo or more members of the same family Rare deleteriousgerminal mutations in the cell cycle regulators CDKN2Aand cyclin dependent-kinase 4 (CDK4) have been shown toconfer a high cutaneous malignant melanoma risk [35 36]Additionally the frequent allelic germinal variants (MC1RASIP MTAP MATP and Casp8) have been identified aslow-risk susceptibility genes or as modifiers of high-riskgenes [37 38] Recently a germinal mutation in the mastergene of melanocyte homeostasis microphthalmia-associatedtranscription factor (MITF) has been identified and shownto increase the risk of developing melanoma [39ndash42] Finallysubjects who have had a melanoma have an approximately9-fold increased risk of developing subsequent melanomacompared with the general population [43]

132 Acquired Risk Factors Epidemiologic studies revealedthat sun exposure is the major known environmental factorassociated with development of melanoma According to theworld health organization (WHO) changes in sun exposurehabits and attitudes that often resulted in excessive UVexposure were recorded in the recent decades and is themaincause of the increase in skin cancer number

Excessive sunlight exposure associated with sunburnespecially in childhood and early adolescence years starts thetransformation of benign melanocytes into a malignant phe-notype Results fromNoonan et al who used a mouse modelexpressing HGF derived by the metallothionein promoterconstitute proof-of-concept [44] A single UV dose deliveredat the neonatal stage was sufficient to induce melanoma inHGFSF-transgenic mice after a relatively short latent periodand with high cumulative incidence Noteworthy the doseroughly administrated to mice in this study corresponds toa sunburning dose of natural sunlight at midlatitudes inmidsummer No melanoma was detected when UVR wasdelivered at the adult stage

Furthermore one personrsquos risk of developing melanomadoubles if they have more than five sunburns at any age Themajority of melanomas are located on intermittently exposed

4 Scientifica

body sites such as the trunk inmen and lower limbs inwomen[45]

Sunrsquos ultraviolet rays are divided into wavelength rangesidentified as UVA (315 to 400 nm) UVB (280 to 315 nm) andUVC (100 to 280 nm) UVC is filtered by the stratosphericozone layer and in theory it does not reach earthrsquos surfaceOfthe UV solar radiation that does reach the earth UVA (95)and UVB (5) promote deleterious effects on proteins andnucleic acids UVB is thought to be more carcinogenic thanUVA by inducing the formation of cyclobutane pyrimidinedimers (CPD) and 6-pyrimidine 4-pyrimidone photoprod-ucts [46] while UVA mainly produces oxidative stress [47]The frequency of CPD generation is three times higher andthey are less efficiently repaired than 6-4 photoproducts TheUVB-induced lesions generate typical genetic mutations Cto T and CC to TT transitions called the ldquoUVB signaturemutationsrdquo Both UVA and UVB can also trigger DNAdamage through oxidative stress and indirectly damage DNAand cause genetic alterations Experiments using animalmodels showed that UVB but not UVA was melanomagenic[22 48]

Therefore UVR may contribute to melanoma develop-ment through combined genotoxic and mitogenic effectsin melanocytes DNA repair-deficient xeroderma pigmento-sum A (Xpa) mice display higher melanocyte proliferationinduction by UVB exposure [23] and individuals with XPmutations display a greatly elevated incidence of skin cancersincluding melanoma [49] thereby indicating that DNA-repair genes play an active role in melanoma initiation DNArepair genes are also involved in melanoma progression byconferring a sort of metastatic genome stabilization duringthe metastatic process [50] The molecular mechanism bywhich these genes are controlled are poorly elucidated yetfunctional genomic experiments unveiled that the mastergene ofmelanocyte homeostasisMITF controls the transcrip-tion of several genes involved in DNA replication and repair[30]

Together these observations point to the importanceof effective DNA damage repair and genomic stability inmelanocyte transformation and melanoma initiation

The absence of UV ldquosignaturerdquo mutations in genes rele-vant to melanoma such as BRAF NRAS CDKN2A or evenp53 which harbor typical UV signature in nonmelanomaskin cancers [51] questioned for years the role of UVR inmelanoma disease Numerous recent publications solved thismystery using ldquodeep sequencing approachesrdquo by showingfrequent UV signature mutation in the genome of differentmelanoma types thereby providing the first genomic evi-dence for a direct mutagenic role of UV light in melanomapathogenesis

In a first study the authors sequenced the coding exonsof 518 kinases in several cancers including melanoma andgenome sequencing of a malignant melanoma and a lym-phoblastoid cell line from the same person revealed thatthe dominant mutational signature was CgtT transversionreflecting DNA damage due to ultraviolet light exposure[52 53] Of the 33345 somatic base substitutions reportedby Pleasance et al almost 25000 were CgtT changes and360 were CCgtTT changes Additionally a high frequency

of GgtT substitutions that might reflect transversion ensuingoxidative DNA damage has been detected [54] A highfrequency of CgtT transversions was nevertheless also iden-tified in non-sun-related cancers such as glioma gastric orcolorectal cancers More evidence came from three otherstudies in which they compared the mutational status ofmelanomas from different body areas such as melanomasfrom the trunk versus sun-shielded acral mucosal and uvealmelanomas [55ndash57] The rate of point mutations was thelowest in primaries from non-ultraviolet-exposed hairlessskin of the extremities (3 and 14 per megabase (Mb) ofgenome) The mutational rate was scored intermediate inthose originating from hair-bearing skin of the trunk (5ndash55 per Mb) and highest in a patient with chronic sunexposure (111 per Mb) [55] Moreover these studies pointedout to mutations affecting the biological function of proteinssuch as PREX2 (phosphatidylinositol-345-trisphosphate-dependent Rac exchange factor 2)-a PTEN-interacting pro-tein and negative regulator of PTEN which is mutated in14 [55] PPP6C encoding a serinethreonine phosphatasemutated in 9ndash124 and RAC1 encoding a GTPase of theRAS superfamily of small GTP-binding proteins mutated in5ndash92 [56 57] of sun-exposedmelanoma cases NoteworthyPPP6C mutations were more frequent in melanomas thatwere mutated for both BRAF and NRAS while RAC1 muta-tions were more frequent in melanomas that were wild-typefor both BRAF and NRAS [56 57] Therefore these studiesfirmly implicatedUV irradiation inmelanomas and providedpotential new therapeutic options

2 Melanoma Disease From Molecular Biologyto Clinic

Tumor growth is the result of genetic andor epigeneticalterations in key genes (ldquodriverrdquo genes) controlling processessuch as proliferation apoptosis senescence and responseto DNA damage These changes lead to the synthesis ofmodifiedactivated (for oncogenes) or hypofunctionalabsent(for tumor suppressor genes or DNA repair genes) proteinsChanges in the stoichiometry and normal biological behaviorof these proteins within the cells will promote an accelerationof the tumor progression At the initiation stage the geneticalteration can be germline Aswe know that the prognosis of apatient is closely linked to its early diagnosis identification ofcancer predisposing genes is crucial to identify and monitorat-risk patients This chapter provides an overview of the keymolecular proteins and associated pathways implicated in theacquisition of the malignant melanoma phenotype

21 Melanoma Susceptibility Genes Historically high-riskgermline mutations in cancer predisposing genes (oncogenesor tumor suppressor genes) were discovered through linkageanalysis in large pedigrees showing Mendelian-like modeof inheritance During the last five years genome-wideassociation studies (GWAS) have identified common SNPsassociated with a low risk of developing cancers includingmelanoma [58] However formany cancers as for other com-plex diseases and human traits the known loci explain only

Scientifica 5

relatively small fractions of the total genetic variance andthe identification of genetic variants with low allele frequencyconferring a moderate-risk of cancer through these classicalapproaches is much more challenging Therefore mutationscreening of candidate genes (or of the entire exome nowachievable thanks to the latest improvement in sequencingtechnologies) in carefully selected at-risk patients coupledwith appropriate tools to assess the pathogenicity of thegenetic variants (in silico and in vitro assays) is an importantcomplementary approach of choice to identify the missingheritability

Recent studies have demonstrated that common diseasescan be due to dysfunctional variants with a wide spectrumof allele frequencies ranging from rare to common Thisis the case for example in breast cancer where high-riskmutations inBRCA1BRCA2 PTEN andTP53 intermediate-risk variants in genes of the DNA repair pathways (ATMCHEK2 BRIP1 and PALB2) and low-risk SNPs (in FGFR2TOX3 CASP8MAP3K1 and LSP1) have been identified [59]

Until very recently these rare sometimes privatemoderate-risk susceptibility alleles have been identifiedthrough resequencing of candidate genes selected on thebasis of biological plausibility A candidate gene approachhas been applied to identify the MITF E318K mutationIn this particular case the fact that two studies reportedthe same recurrent MITF mutation in independent patientseriespopulations using an agnostic genome-wide approach(massive parallel exome sequencing approach plus genotyp-ing in larger melanoma series familial CMM where multipleprimaries often occur) reinforces the notion of the existenceof variants with low minor allele frequency that could havesubstantial effects (Figure 2)

211 Genes with High Penetrance Rare alleles of CDKN2Aand CDK4 genes have been identified in familial formsof melanoma among patients who have had melanomaMutations or deletions in these genes confer an elevatedrisk of developing melanoma These genes are involved incell cycle arrest and cellular senescence Their importance isrevealed by the fact that germline and somatic mutations inCDKN2A and CDK4 directly or in their associated signalingpathways are almost invariably found in melanomas

The CDKN2A Locus CDKN2A (Cyclin-Dependent KinaseInhibitor 2A) is located at the 9p21 locus and encodes twotumor suppressor proteins p16INK4a (Inhibitor of Kinase a)and p14ARF (Alternative Reading Frame) p16INK4A is tran-scribed from exons 1120572 2 and 3 with exon 3 encoding onlyfour amino acids p14ARF is encoded by an alternative exon 1(1120573) spliced to CDKN2A exon 2 in an alternate reading frame(ARF) The p16INK4a and p14ARF transcripts are translatedin different reading frames thus the two proteins have nohomology at the amino acid level [60] In familial melanomawhich represents 8 to 12 of all melanomas [61] germlinemutations in the CDKN2A gene are found in 20ndash40of cases[62] These mutations can affect p16INK4a p14ARF or bothproteins p16INK4a interacts specifically with both CDK4 andCDK6 and blocks their association with D-type complexes

[63] Thus the loss of function of p16INK4a promotes CDK4and CDK6 activation resulting in hyperphosphorylation ofpRB and the activation of the transcription factor E2F1E2F1 mediates the transcription of S phase promoting genesthereby promoting cell proliferation

Evidence of a role for p16INK4a in human melanomaincludes frequent somatic genetic and epigenetic alterationsin human melanoma samples Indeed p16INK4a gene is lostin 50 of melanoma cases inactivated by point mutations inapproximately 9 of tumors or inactivated by methylationof its promoter in about 10 of melanoma cases [64]p16INK4a loss of function promotes senescence bypass andmelanocyte immortalization [65 66] Senescence is a pro-gram that represents a potent barrier against tumorigenesisby preventing the proliferation of cells at risk for neoplasictransformation The reintroduction of a functional copy ofp16INK4a in melanoma cell lines induced a significant changein morphology associated with dendricity a parameter ofdifferentiation and decreased cell growth [67]

The collaboration of oncogenic NRASQ61K and p16INK4a

loss of expression is sufficient to cause melanoma formationin mice supporting the critical role of p16INK4a in melanomaaetiology [68]

Although p16INK4a was thought to be the predominanttumor suppressor at 9p21 observations from melanoma-prone families in which exon 1120573 germline deletion or muta-tion in either the coding region or splice donor site of thisexon have been reported supporting a p16INK4a-independenttumor suppressor role for p14ARF [69ndash71] A comprehensiveanalysis of the pattern of genetic and epigenetic alterations tothe p16INK4a and p14ARF tumor suppressor loci in melanomarevealed that p14ARF is frequently inactivated [72] Sup-porting the role of p14ARF in melanomagenesis murinemelanoma models demonstrated that specific inactivationof p14ARF (p19ARF in mouse) enhanced melanoma devel-opment [73 74] Although p14ARF is mainly known tofunction by preventing p53 degradation by the E3 ubiquitin-protein ligase MDM2 (Mouse Double Minute 2) in mousecontext melanomagenesis in ARF-depleted background isp53-independent [73 74]

Consistently activating mutations of BRAF and loss offunctional p16INK4a and p14ARF were detected in themajorityof melanomas [75]

CDK4 Germline mutations in the gene encoding cyclindependent kinase 4 (CDK4A) have been identified in avery small percentage of familial melanoma [36 76] Themutation of arginine at position 24 into cysteine (CDK4R24C)or histidine (CDK4R24C) renders the protein insensitive toregulation by p16INK4a but preserves interaction betweenCDK4 and cyclin D1 leading to constitutive activation of thecomplex and aberrant proliferation through retinoblastomaprotein inactivation and E2F activation CDK4R24C facilitatestumorigenesis of melanocytes transplanted into nude miceand causes escape from cellular senescence [77] Mousemodels show that the CDK4R24C form enhances melanomapenetrance in cooperationwith the oncogenicHRasG12V [78]

6 Scientifica

Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

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Scientifica 3

activation via the 120572MSHcAMP cascade led to an increase ofproliferation upon the MEKERK signaling pathway activa-tion [26] This report was the beginning of what is now thebiggest focus in the melanocyte field the role of BRAF inmelanoma disease Activation of the ERK and PI3K signalingpathways are also associated with a survival program Indeedconsidering the key role of melanocytes in protecting ourbody against the noxious effect of UVR melanocytes havedeveloped potent antiapoptoticmechanisms involved in theirresistance to the UVR-induced DNA damage and cell deathIn this context MITF whose expression is increased uponUVR controls the expression of antiapoptotic genes such asBCL2 [27] BCL2A1 [28] andML-IAP [29] and several genesinvolved in DNA repair [30]

Exposure of human skinto sunlight has positive healtheffects in its ability to boost the bodyrsquos vitamin D supplywhich is essential for overall body health The avoidanceof all direct sun exposure increases the risk of vitamin Ddeficiency which can have serious consequences amongwhich increased risks of deadly cancers [31 32] Howeverfrequent and intense exposure to UV radiation sunlightespecially in childhood is the major environmental riskfactor for melanoma development

These observations illustrate how important is the del-icate balance between differentiation and proliferationsur-vival of melanocytes

13 Melanoma Disease Cutaneous metastatic melanoma(CMM) deriving from melanocyte transformation in 75 ofthe cases and from preexisting nevi in 25 of the cases isthe most deadly form of skin cancer Melanoma accounts forless than 5 of skin cancers but is responsible for 80 ofskin cancer related deathsMelanoma incidence in Caucasianpopulation has increased dramatically worldwide during thepast several decades Melanoma is a cancer with a relativelygood prognosis when diagnosed early at a cutaneous local-ized stage Patients with stage 0I thin lesions can usuallybe cured with surgical excision Their 5-year survival rateranges from 90 to 100 However the prognosis worsensthe deeper the lesion extends beneath the skin becauseof melanomarsquos propensity to invade and to metastasizeIndividuals with thick melanomas have an increased riskto develop lymph node and visceral metastases Metastaticmelanoma cannot be completely removed by surgery andmetastatic cells display extreme resistance to all types oftreatment Patientswithmetastaticmelanomas have amediansurvival rate that typically ranges from six to ten months

Melanomagenesis is a complex phenomenon in whichenvironmental genetic and host factors play a role Datafrom the clinic epidemiology andmore recently from geneticreveal that melanomas are heterogeneous tumors harboringvarious genetic alterations developing at different body sitesand on sun-exposed and non sun-exposed regions suggest-ing that melanoma arises from divergent causal pathwaysCurtin et al proposed a molecular classification based onthe sites where the melanoma occurs the genetic alterationsand the sun exposure history [33] Mutations in BRAF weresignificantly more common in melanomas located in areas

without chronic sun-induced damage Melanomas arising inchronically sun-damaged skin mucosal surfaces and acralskin were characterized by wild-type BRAF and wild-typeNRAS but exhibited alterations in KIT

131 The Constitutional Risk Factors We are not all equal infront of the sun Major risk factors include skin phototypeand skin reaction to sun exposure according to the phototypea high number of nevidysplasic nevi and a personal andfamilial history of melanoma

Subjects with redblond hair blue eyes fair skin anddeveloping sunburns have higher melanoma risk than sub-jects with brown haireyes and skin that tan easily Theformer produce little or no eumelanin and are thereforemuch less protected from the noxious effects of UV radiationSubjects with gt50 nevi or having at least five atypical molesexhibit fivefold higher risk of developing melanoma Thepresence of large congenital nevi is also a melanoma riskfactor [34] Finally approximately 10ofmelanomas occur ina familial context defined by at least two melanomas withintwo or more members of the same family Rare deleteriousgerminal mutations in the cell cycle regulators CDKN2Aand cyclin dependent-kinase 4 (CDK4) have been shown toconfer a high cutaneous malignant melanoma risk [35 36]Additionally the frequent allelic germinal variants (MC1RASIP MTAP MATP and Casp8) have been identified aslow-risk susceptibility genes or as modifiers of high-riskgenes [37 38] Recently a germinal mutation in the mastergene of melanocyte homeostasis microphthalmia-associatedtranscription factor (MITF) has been identified and shownto increase the risk of developing melanoma [39ndash42] Finallysubjects who have had a melanoma have an approximately9-fold increased risk of developing subsequent melanomacompared with the general population [43]

132 Acquired Risk Factors Epidemiologic studies revealedthat sun exposure is the major known environmental factorassociated with development of melanoma According to theworld health organization (WHO) changes in sun exposurehabits and attitudes that often resulted in excessive UVexposure were recorded in the recent decades and is themaincause of the increase in skin cancer number

Excessive sunlight exposure associated with sunburnespecially in childhood and early adolescence years starts thetransformation of benign melanocytes into a malignant phe-notype Results fromNoonan et al who used a mouse modelexpressing HGF derived by the metallothionein promoterconstitute proof-of-concept [44] A single UV dose deliveredat the neonatal stage was sufficient to induce melanoma inHGFSF-transgenic mice after a relatively short latent periodand with high cumulative incidence Noteworthy the doseroughly administrated to mice in this study corresponds toa sunburning dose of natural sunlight at midlatitudes inmidsummer No melanoma was detected when UVR wasdelivered at the adult stage

Furthermore one personrsquos risk of developing melanomadoubles if they have more than five sunburns at any age Themajority of melanomas are located on intermittently exposed

4 Scientifica

body sites such as the trunk inmen and lower limbs inwomen[45]

Sunrsquos ultraviolet rays are divided into wavelength rangesidentified as UVA (315 to 400 nm) UVB (280 to 315 nm) andUVC (100 to 280 nm) UVC is filtered by the stratosphericozone layer and in theory it does not reach earthrsquos surfaceOfthe UV solar radiation that does reach the earth UVA (95)and UVB (5) promote deleterious effects on proteins andnucleic acids UVB is thought to be more carcinogenic thanUVA by inducing the formation of cyclobutane pyrimidinedimers (CPD) and 6-pyrimidine 4-pyrimidone photoprod-ucts [46] while UVA mainly produces oxidative stress [47]The frequency of CPD generation is three times higher andthey are less efficiently repaired than 6-4 photoproducts TheUVB-induced lesions generate typical genetic mutations Cto T and CC to TT transitions called the ldquoUVB signaturemutationsrdquo Both UVA and UVB can also trigger DNAdamage through oxidative stress and indirectly damage DNAand cause genetic alterations Experiments using animalmodels showed that UVB but not UVA was melanomagenic[22 48]

Therefore UVR may contribute to melanoma develop-ment through combined genotoxic and mitogenic effectsin melanocytes DNA repair-deficient xeroderma pigmento-sum A (Xpa) mice display higher melanocyte proliferationinduction by UVB exposure [23] and individuals with XPmutations display a greatly elevated incidence of skin cancersincluding melanoma [49] thereby indicating that DNA-repair genes play an active role in melanoma initiation DNArepair genes are also involved in melanoma progression byconferring a sort of metastatic genome stabilization duringthe metastatic process [50] The molecular mechanism bywhich these genes are controlled are poorly elucidated yetfunctional genomic experiments unveiled that the mastergene ofmelanocyte homeostasisMITF controls the transcrip-tion of several genes involved in DNA replication and repair[30]

Together these observations point to the importanceof effective DNA damage repair and genomic stability inmelanocyte transformation and melanoma initiation

The absence of UV ldquosignaturerdquo mutations in genes rele-vant to melanoma such as BRAF NRAS CDKN2A or evenp53 which harbor typical UV signature in nonmelanomaskin cancers [51] questioned for years the role of UVR inmelanoma disease Numerous recent publications solved thismystery using ldquodeep sequencing approachesrdquo by showingfrequent UV signature mutation in the genome of differentmelanoma types thereby providing the first genomic evi-dence for a direct mutagenic role of UV light in melanomapathogenesis

In a first study the authors sequenced the coding exonsof 518 kinases in several cancers including melanoma andgenome sequencing of a malignant melanoma and a lym-phoblastoid cell line from the same person revealed thatthe dominant mutational signature was CgtT transversionreflecting DNA damage due to ultraviolet light exposure[52 53] Of the 33345 somatic base substitutions reportedby Pleasance et al almost 25000 were CgtT changes and360 were CCgtTT changes Additionally a high frequency

of GgtT substitutions that might reflect transversion ensuingoxidative DNA damage has been detected [54] A highfrequency of CgtT transversions was nevertheless also iden-tified in non-sun-related cancers such as glioma gastric orcolorectal cancers More evidence came from three otherstudies in which they compared the mutational status ofmelanomas from different body areas such as melanomasfrom the trunk versus sun-shielded acral mucosal and uvealmelanomas [55ndash57] The rate of point mutations was thelowest in primaries from non-ultraviolet-exposed hairlessskin of the extremities (3 and 14 per megabase (Mb) ofgenome) The mutational rate was scored intermediate inthose originating from hair-bearing skin of the trunk (5ndash55 per Mb) and highest in a patient with chronic sunexposure (111 per Mb) [55] Moreover these studies pointedout to mutations affecting the biological function of proteinssuch as PREX2 (phosphatidylinositol-345-trisphosphate-dependent Rac exchange factor 2)-a PTEN-interacting pro-tein and negative regulator of PTEN which is mutated in14 [55] PPP6C encoding a serinethreonine phosphatasemutated in 9ndash124 and RAC1 encoding a GTPase of theRAS superfamily of small GTP-binding proteins mutated in5ndash92 [56 57] of sun-exposedmelanoma cases NoteworthyPPP6C mutations were more frequent in melanomas thatwere mutated for both BRAF and NRAS while RAC1 muta-tions were more frequent in melanomas that were wild-typefor both BRAF and NRAS [56 57] Therefore these studiesfirmly implicatedUV irradiation inmelanomas and providedpotential new therapeutic options

2 Melanoma Disease From Molecular Biologyto Clinic

Tumor growth is the result of genetic andor epigeneticalterations in key genes (ldquodriverrdquo genes) controlling processessuch as proliferation apoptosis senescence and responseto DNA damage These changes lead to the synthesis ofmodifiedactivated (for oncogenes) or hypofunctionalabsent(for tumor suppressor genes or DNA repair genes) proteinsChanges in the stoichiometry and normal biological behaviorof these proteins within the cells will promote an accelerationof the tumor progression At the initiation stage the geneticalteration can be germline Aswe know that the prognosis of apatient is closely linked to its early diagnosis identification ofcancer predisposing genes is crucial to identify and monitorat-risk patients This chapter provides an overview of the keymolecular proteins and associated pathways implicated in theacquisition of the malignant melanoma phenotype

21 Melanoma Susceptibility Genes Historically high-riskgermline mutations in cancer predisposing genes (oncogenesor tumor suppressor genes) were discovered through linkageanalysis in large pedigrees showing Mendelian-like modeof inheritance During the last five years genome-wideassociation studies (GWAS) have identified common SNPsassociated with a low risk of developing cancers includingmelanoma [58] However formany cancers as for other com-plex diseases and human traits the known loci explain only

Scientifica 5

relatively small fractions of the total genetic variance andthe identification of genetic variants with low allele frequencyconferring a moderate-risk of cancer through these classicalapproaches is much more challenging Therefore mutationscreening of candidate genes (or of the entire exome nowachievable thanks to the latest improvement in sequencingtechnologies) in carefully selected at-risk patients coupledwith appropriate tools to assess the pathogenicity of thegenetic variants (in silico and in vitro assays) is an importantcomplementary approach of choice to identify the missingheritability

Recent studies have demonstrated that common diseasescan be due to dysfunctional variants with a wide spectrumof allele frequencies ranging from rare to common Thisis the case for example in breast cancer where high-riskmutations inBRCA1BRCA2 PTEN andTP53 intermediate-risk variants in genes of the DNA repair pathways (ATMCHEK2 BRIP1 and PALB2) and low-risk SNPs (in FGFR2TOX3 CASP8MAP3K1 and LSP1) have been identified [59]

Until very recently these rare sometimes privatemoderate-risk susceptibility alleles have been identifiedthrough resequencing of candidate genes selected on thebasis of biological plausibility A candidate gene approachhas been applied to identify the MITF E318K mutationIn this particular case the fact that two studies reportedthe same recurrent MITF mutation in independent patientseriespopulations using an agnostic genome-wide approach(massive parallel exome sequencing approach plus genotyp-ing in larger melanoma series familial CMM where multipleprimaries often occur) reinforces the notion of the existenceof variants with low minor allele frequency that could havesubstantial effects (Figure 2)

211 Genes with High Penetrance Rare alleles of CDKN2Aand CDK4 genes have been identified in familial formsof melanoma among patients who have had melanomaMutations or deletions in these genes confer an elevatedrisk of developing melanoma These genes are involved incell cycle arrest and cellular senescence Their importance isrevealed by the fact that germline and somatic mutations inCDKN2A and CDK4 directly or in their associated signalingpathways are almost invariably found in melanomas

The CDKN2A Locus CDKN2A (Cyclin-Dependent KinaseInhibitor 2A) is located at the 9p21 locus and encodes twotumor suppressor proteins p16INK4a (Inhibitor of Kinase a)and p14ARF (Alternative Reading Frame) p16INK4A is tran-scribed from exons 1120572 2 and 3 with exon 3 encoding onlyfour amino acids p14ARF is encoded by an alternative exon 1(1120573) spliced to CDKN2A exon 2 in an alternate reading frame(ARF) The p16INK4a and p14ARF transcripts are translatedin different reading frames thus the two proteins have nohomology at the amino acid level [60] In familial melanomawhich represents 8 to 12 of all melanomas [61] germlinemutations in the CDKN2A gene are found in 20ndash40of cases[62] These mutations can affect p16INK4a p14ARF or bothproteins p16INK4a interacts specifically with both CDK4 andCDK6 and blocks their association with D-type complexes

[63] Thus the loss of function of p16INK4a promotes CDK4and CDK6 activation resulting in hyperphosphorylation ofpRB and the activation of the transcription factor E2F1E2F1 mediates the transcription of S phase promoting genesthereby promoting cell proliferation

Evidence of a role for p16INK4a in human melanomaincludes frequent somatic genetic and epigenetic alterationsin human melanoma samples Indeed p16INK4a gene is lostin 50 of melanoma cases inactivated by point mutations inapproximately 9 of tumors or inactivated by methylationof its promoter in about 10 of melanoma cases [64]p16INK4a loss of function promotes senescence bypass andmelanocyte immortalization [65 66] Senescence is a pro-gram that represents a potent barrier against tumorigenesisby preventing the proliferation of cells at risk for neoplasictransformation The reintroduction of a functional copy ofp16INK4a in melanoma cell lines induced a significant changein morphology associated with dendricity a parameter ofdifferentiation and decreased cell growth [67]

The collaboration of oncogenic NRASQ61K and p16INK4a

loss of expression is sufficient to cause melanoma formationin mice supporting the critical role of p16INK4a in melanomaaetiology [68]

Although p16INK4a was thought to be the predominanttumor suppressor at 9p21 observations from melanoma-prone families in which exon 1120573 germline deletion or muta-tion in either the coding region or splice donor site of thisexon have been reported supporting a p16INK4a-independenttumor suppressor role for p14ARF [69ndash71] A comprehensiveanalysis of the pattern of genetic and epigenetic alterations tothe p16INK4a and p14ARF tumor suppressor loci in melanomarevealed that p14ARF is frequently inactivated [72] Sup-porting the role of p14ARF in melanomagenesis murinemelanoma models demonstrated that specific inactivationof p14ARF (p19ARF in mouse) enhanced melanoma devel-opment [73 74] Although p14ARF is mainly known tofunction by preventing p53 degradation by the E3 ubiquitin-protein ligase MDM2 (Mouse Double Minute 2) in mousecontext melanomagenesis in ARF-depleted background isp53-independent [73 74]

Consistently activating mutations of BRAF and loss offunctional p16INK4a and p14ARF were detected in themajorityof melanomas [75]

CDK4 Germline mutations in the gene encoding cyclindependent kinase 4 (CDK4A) have been identified in avery small percentage of familial melanoma [36 76] Themutation of arginine at position 24 into cysteine (CDK4R24C)or histidine (CDK4R24C) renders the protein insensitive toregulation by p16INK4a but preserves interaction betweenCDK4 and cyclin D1 leading to constitutive activation of thecomplex and aberrant proliferation through retinoblastomaprotein inactivation and E2F activation CDK4R24C facilitatestumorigenesis of melanocytes transplanted into nude miceand causes escape from cellular senescence [77] Mousemodels show that the CDK4R24C form enhances melanomapenetrance in cooperationwith the oncogenicHRasG12V [78]

6 Scientifica

Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Scientifica

body sites such as the trunk inmen and lower limbs inwomen[45]

Sunrsquos ultraviolet rays are divided into wavelength rangesidentified as UVA (315 to 400 nm) UVB (280 to 315 nm) andUVC (100 to 280 nm) UVC is filtered by the stratosphericozone layer and in theory it does not reach earthrsquos surfaceOfthe UV solar radiation that does reach the earth UVA (95)and UVB (5) promote deleterious effects on proteins andnucleic acids UVB is thought to be more carcinogenic thanUVA by inducing the formation of cyclobutane pyrimidinedimers (CPD) and 6-pyrimidine 4-pyrimidone photoprod-ucts [46] while UVA mainly produces oxidative stress [47]The frequency of CPD generation is three times higher andthey are less efficiently repaired than 6-4 photoproducts TheUVB-induced lesions generate typical genetic mutations Cto T and CC to TT transitions called the ldquoUVB signaturemutationsrdquo Both UVA and UVB can also trigger DNAdamage through oxidative stress and indirectly damage DNAand cause genetic alterations Experiments using animalmodels showed that UVB but not UVA was melanomagenic[22 48]

Therefore UVR may contribute to melanoma develop-ment through combined genotoxic and mitogenic effectsin melanocytes DNA repair-deficient xeroderma pigmento-sum A (Xpa) mice display higher melanocyte proliferationinduction by UVB exposure [23] and individuals with XPmutations display a greatly elevated incidence of skin cancersincluding melanoma [49] thereby indicating that DNA-repair genes play an active role in melanoma initiation DNArepair genes are also involved in melanoma progression byconferring a sort of metastatic genome stabilization duringthe metastatic process [50] The molecular mechanism bywhich these genes are controlled are poorly elucidated yetfunctional genomic experiments unveiled that the mastergene ofmelanocyte homeostasisMITF controls the transcrip-tion of several genes involved in DNA replication and repair[30]

Together these observations point to the importanceof effective DNA damage repair and genomic stability inmelanocyte transformation and melanoma initiation

The absence of UV ldquosignaturerdquo mutations in genes rele-vant to melanoma such as BRAF NRAS CDKN2A or evenp53 which harbor typical UV signature in nonmelanomaskin cancers [51] questioned for years the role of UVR inmelanoma disease Numerous recent publications solved thismystery using ldquodeep sequencing approachesrdquo by showingfrequent UV signature mutation in the genome of differentmelanoma types thereby providing the first genomic evi-dence for a direct mutagenic role of UV light in melanomapathogenesis

In a first study the authors sequenced the coding exonsof 518 kinases in several cancers including melanoma andgenome sequencing of a malignant melanoma and a lym-phoblastoid cell line from the same person revealed thatthe dominant mutational signature was CgtT transversionreflecting DNA damage due to ultraviolet light exposure[52 53] Of the 33345 somatic base substitutions reportedby Pleasance et al almost 25000 were CgtT changes and360 were CCgtTT changes Additionally a high frequency

of GgtT substitutions that might reflect transversion ensuingoxidative DNA damage has been detected [54] A highfrequency of CgtT transversions was nevertheless also iden-tified in non-sun-related cancers such as glioma gastric orcolorectal cancers More evidence came from three otherstudies in which they compared the mutational status ofmelanomas from different body areas such as melanomasfrom the trunk versus sun-shielded acral mucosal and uvealmelanomas [55ndash57] The rate of point mutations was thelowest in primaries from non-ultraviolet-exposed hairlessskin of the extremities (3 and 14 per megabase (Mb) ofgenome) The mutational rate was scored intermediate inthose originating from hair-bearing skin of the trunk (5ndash55 per Mb) and highest in a patient with chronic sunexposure (111 per Mb) [55] Moreover these studies pointedout to mutations affecting the biological function of proteinssuch as PREX2 (phosphatidylinositol-345-trisphosphate-dependent Rac exchange factor 2)-a PTEN-interacting pro-tein and negative regulator of PTEN which is mutated in14 [55] PPP6C encoding a serinethreonine phosphatasemutated in 9ndash124 and RAC1 encoding a GTPase of theRAS superfamily of small GTP-binding proteins mutated in5ndash92 [56 57] of sun-exposedmelanoma cases NoteworthyPPP6C mutations were more frequent in melanomas thatwere mutated for both BRAF and NRAS while RAC1 muta-tions were more frequent in melanomas that were wild-typefor both BRAF and NRAS [56 57] Therefore these studiesfirmly implicatedUV irradiation inmelanomas and providedpotential new therapeutic options

2 Melanoma Disease From Molecular Biologyto Clinic

Tumor growth is the result of genetic andor epigeneticalterations in key genes (ldquodriverrdquo genes) controlling processessuch as proliferation apoptosis senescence and responseto DNA damage These changes lead to the synthesis ofmodifiedactivated (for oncogenes) or hypofunctionalabsent(for tumor suppressor genes or DNA repair genes) proteinsChanges in the stoichiometry and normal biological behaviorof these proteins within the cells will promote an accelerationof the tumor progression At the initiation stage the geneticalteration can be germline Aswe know that the prognosis of apatient is closely linked to its early diagnosis identification ofcancer predisposing genes is crucial to identify and monitorat-risk patients This chapter provides an overview of the keymolecular proteins and associated pathways implicated in theacquisition of the malignant melanoma phenotype

21 Melanoma Susceptibility Genes Historically high-riskgermline mutations in cancer predisposing genes (oncogenesor tumor suppressor genes) were discovered through linkageanalysis in large pedigrees showing Mendelian-like modeof inheritance During the last five years genome-wideassociation studies (GWAS) have identified common SNPsassociated with a low risk of developing cancers includingmelanoma [58] However formany cancers as for other com-plex diseases and human traits the known loci explain only

Scientifica 5

relatively small fractions of the total genetic variance andthe identification of genetic variants with low allele frequencyconferring a moderate-risk of cancer through these classicalapproaches is much more challenging Therefore mutationscreening of candidate genes (or of the entire exome nowachievable thanks to the latest improvement in sequencingtechnologies) in carefully selected at-risk patients coupledwith appropriate tools to assess the pathogenicity of thegenetic variants (in silico and in vitro assays) is an importantcomplementary approach of choice to identify the missingheritability

Recent studies have demonstrated that common diseasescan be due to dysfunctional variants with a wide spectrumof allele frequencies ranging from rare to common Thisis the case for example in breast cancer where high-riskmutations inBRCA1BRCA2 PTEN andTP53 intermediate-risk variants in genes of the DNA repair pathways (ATMCHEK2 BRIP1 and PALB2) and low-risk SNPs (in FGFR2TOX3 CASP8MAP3K1 and LSP1) have been identified [59]

Until very recently these rare sometimes privatemoderate-risk susceptibility alleles have been identifiedthrough resequencing of candidate genes selected on thebasis of biological plausibility A candidate gene approachhas been applied to identify the MITF E318K mutationIn this particular case the fact that two studies reportedthe same recurrent MITF mutation in independent patientseriespopulations using an agnostic genome-wide approach(massive parallel exome sequencing approach plus genotyp-ing in larger melanoma series familial CMM where multipleprimaries often occur) reinforces the notion of the existenceof variants with low minor allele frequency that could havesubstantial effects (Figure 2)

211 Genes with High Penetrance Rare alleles of CDKN2Aand CDK4 genes have been identified in familial formsof melanoma among patients who have had melanomaMutations or deletions in these genes confer an elevatedrisk of developing melanoma These genes are involved incell cycle arrest and cellular senescence Their importance isrevealed by the fact that germline and somatic mutations inCDKN2A and CDK4 directly or in their associated signalingpathways are almost invariably found in melanomas

The CDKN2A Locus CDKN2A (Cyclin-Dependent KinaseInhibitor 2A) is located at the 9p21 locus and encodes twotumor suppressor proteins p16INK4a (Inhibitor of Kinase a)and p14ARF (Alternative Reading Frame) p16INK4A is tran-scribed from exons 1120572 2 and 3 with exon 3 encoding onlyfour amino acids p14ARF is encoded by an alternative exon 1(1120573) spliced to CDKN2A exon 2 in an alternate reading frame(ARF) The p16INK4a and p14ARF transcripts are translatedin different reading frames thus the two proteins have nohomology at the amino acid level [60] In familial melanomawhich represents 8 to 12 of all melanomas [61] germlinemutations in the CDKN2A gene are found in 20ndash40of cases[62] These mutations can affect p16INK4a p14ARF or bothproteins p16INK4a interacts specifically with both CDK4 andCDK6 and blocks their association with D-type complexes

[63] Thus the loss of function of p16INK4a promotes CDK4and CDK6 activation resulting in hyperphosphorylation ofpRB and the activation of the transcription factor E2F1E2F1 mediates the transcription of S phase promoting genesthereby promoting cell proliferation

Evidence of a role for p16INK4a in human melanomaincludes frequent somatic genetic and epigenetic alterationsin human melanoma samples Indeed p16INK4a gene is lostin 50 of melanoma cases inactivated by point mutations inapproximately 9 of tumors or inactivated by methylationof its promoter in about 10 of melanoma cases [64]p16INK4a loss of function promotes senescence bypass andmelanocyte immortalization [65 66] Senescence is a pro-gram that represents a potent barrier against tumorigenesisby preventing the proliferation of cells at risk for neoplasictransformation The reintroduction of a functional copy ofp16INK4a in melanoma cell lines induced a significant changein morphology associated with dendricity a parameter ofdifferentiation and decreased cell growth [67]

The collaboration of oncogenic NRASQ61K and p16INK4a

loss of expression is sufficient to cause melanoma formationin mice supporting the critical role of p16INK4a in melanomaaetiology [68]

Although p16INK4a was thought to be the predominanttumor suppressor at 9p21 observations from melanoma-prone families in which exon 1120573 germline deletion or muta-tion in either the coding region or splice donor site of thisexon have been reported supporting a p16INK4a-independenttumor suppressor role for p14ARF [69ndash71] A comprehensiveanalysis of the pattern of genetic and epigenetic alterations tothe p16INK4a and p14ARF tumor suppressor loci in melanomarevealed that p14ARF is frequently inactivated [72] Sup-porting the role of p14ARF in melanomagenesis murinemelanoma models demonstrated that specific inactivationof p14ARF (p19ARF in mouse) enhanced melanoma devel-opment [73 74] Although p14ARF is mainly known tofunction by preventing p53 degradation by the E3 ubiquitin-protein ligase MDM2 (Mouse Double Minute 2) in mousecontext melanomagenesis in ARF-depleted background isp53-independent [73 74]

Consistently activating mutations of BRAF and loss offunctional p16INK4a and p14ARF were detected in themajorityof melanomas [75]

CDK4 Germline mutations in the gene encoding cyclindependent kinase 4 (CDK4A) have been identified in avery small percentage of familial melanoma [36 76] Themutation of arginine at position 24 into cysteine (CDK4R24C)or histidine (CDK4R24C) renders the protein insensitive toregulation by p16INK4a but preserves interaction betweenCDK4 and cyclin D1 leading to constitutive activation of thecomplex and aberrant proliferation through retinoblastomaprotein inactivation and E2F activation CDK4R24C facilitatestumorigenesis of melanocytes transplanted into nude miceand causes escape from cellular senescence [77] Mousemodels show that the CDK4R24C form enhances melanomapenetrance in cooperationwith the oncogenicHRasG12V [78]

6 Scientifica

Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 5

relatively small fractions of the total genetic variance andthe identification of genetic variants with low allele frequencyconferring a moderate-risk of cancer through these classicalapproaches is much more challenging Therefore mutationscreening of candidate genes (or of the entire exome nowachievable thanks to the latest improvement in sequencingtechnologies) in carefully selected at-risk patients coupledwith appropriate tools to assess the pathogenicity of thegenetic variants (in silico and in vitro assays) is an importantcomplementary approach of choice to identify the missingheritability

Recent studies have demonstrated that common diseasescan be due to dysfunctional variants with a wide spectrumof allele frequencies ranging from rare to common Thisis the case for example in breast cancer where high-riskmutations inBRCA1BRCA2 PTEN andTP53 intermediate-risk variants in genes of the DNA repair pathways (ATMCHEK2 BRIP1 and PALB2) and low-risk SNPs (in FGFR2TOX3 CASP8MAP3K1 and LSP1) have been identified [59]

Until very recently these rare sometimes privatemoderate-risk susceptibility alleles have been identifiedthrough resequencing of candidate genes selected on thebasis of biological plausibility A candidate gene approachhas been applied to identify the MITF E318K mutationIn this particular case the fact that two studies reportedthe same recurrent MITF mutation in independent patientseriespopulations using an agnostic genome-wide approach(massive parallel exome sequencing approach plus genotyp-ing in larger melanoma series familial CMM where multipleprimaries often occur) reinforces the notion of the existenceof variants with low minor allele frequency that could havesubstantial effects (Figure 2)

211 Genes with High Penetrance Rare alleles of CDKN2Aand CDK4 genes have been identified in familial formsof melanoma among patients who have had melanomaMutations or deletions in these genes confer an elevatedrisk of developing melanoma These genes are involved incell cycle arrest and cellular senescence Their importance isrevealed by the fact that germline and somatic mutations inCDKN2A and CDK4 directly or in their associated signalingpathways are almost invariably found in melanomas

The CDKN2A Locus CDKN2A (Cyclin-Dependent KinaseInhibitor 2A) is located at the 9p21 locus and encodes twotumor suppressor proteins p16INK4a (Inhibitor of Kinase a)and p14ARF (Alternative Reading Frame) p16INK4A is tran-scribed from exons 1120572 2 and 3 with exon 3 encoding onlyfour amino acids p14ARF is encoded by an alternative exon 1(1120573) spliced to CDKN2A exon 2 in an alternate reading frame(ARF) The p16INK4a and p14ARF transcripts are translatedin different reading frames thus the two proteins have nohomology at the amino acid level [60] In familial melanomawhich represents 8 to 12 of all melanomas [61] germlinemutations in the CDKN2A gene are found in 20ndash40of cases[62] These mutations can affect p16INK4a p14ARF or bothproteins p16INK4a interacts specifically with both CDK4 andCDK6 and blocks their association with D-type complexes

[63] Thus the loss of function of p16INK4a promotes CDK4and CDK6 activation resulting in hyperphosphorylation ofpRB and the activation of the transcription factor E2F1E2F1 mediates the transcription of S phase promoting genesthereby promoting cell proliferation

Evidence of a role for p16INK4a in human melanomaincludes frequent somatic genetic and epigenetic alterationsin human melanoma samples Indeed p16INK4a gene is lostin 50 of melanoma cases inactivated by point mutations inapproximately 9 of tumors or inactivated by methylationof its promoter in about 10 of melanoma cases [64]p16INK4a loss of function promotes senescence bypass andmelanocyte immortalization [65 66] Senescence is a pro-gram that represents a potent barrier against tumorigenesisby preventing the proliferation of cells at risk for neoplasictransformation The reintroduction of a functional copy ofp16INK4a in melanoma cell lines induced a significant changein morphology associated with dendricity a parameter ofdifferentiation and decreased cell growth [67]

The collaboration of oncogenic NRASQ61K and p16INK4a

loss of expression is sufficient to cause melanoma formationin mice supporting the critical role of p16INK4a in melanomaaetiology [68]

Although p16INK4a was thought to be the predominanttumor suppressor at 9p21 observations from melanoma-prone families in which exon 1120573 germline deletion or muta-tion in either the coding region or splice donor site of thisexon have been reported supporting a p16INK4a-independenttumor suppressor role for p14ARF [69ndash71] A comprehensiveanalysis of the pattern of genetic and epigenetic alterations tothe p16INK4a and p14ARF tumor suppressor loci in melanomarevealed that p14ARF is frequently inactivated [72] Sup-porting the role of p14ARF in melanomagenesis murinemelanoma models demonstrated that specific inactivationof p14ARF (p19ARF in mouse) enhanced melanoma devel-opment [73 74] Although p14ARF is mainly known tofunction by preventing p53 degradation by the E3 ubiquitin-protein ligase MDM2 (Mouse Double Minute 2) in mousecontext melanomagenesis in ARF-depleted background isp53-independent [73 74]

Consistently activating mutations of BRAF and loss offunctional p16INK4a and p14ARF were detected in themajorityof melanomas [75]

CDK4 Germline mutations in the gene encoding cyclindependent kinase 4 (CDK4A) have been identified in avery small percentage of familial melanoma [36 76] Themutation of arginine at position 24 into cysteine (CDK4R24C)or histidine (CDK4R24C) renders the protein insensitive toregulation by p16INK4a but preserves interaction betweenCDK4 and cyclin D1 leading to constitutive activation of thecomplex and aberrant proliferation through retinoblastomaprotein inactivation and E2F activation CDK4R24C facilitatestumorigenesis of melanocytes transplanted into nude miceand causes escape from cellular senescence [77] Mousemodels show that the CDK4R24C form enhances melanomapenetrance in cooperationwith the oncogenicHRasG12V [78]

6 Scientifica

Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

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pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

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6 Scientifica

Very rare Rare Weak Common

High

Moderate

Intermediate

Weak

500

30

15

11

Size

effec

t

Minor allele frequency

0001 0005 005

CDKN2ACDK4

MITF

ASIP MTAPCASP8 OCA2MATP Tyr TYRP1MC1R TERT

MC1R

Figure 2 Melanoma susceptibility alleles (adapted fromManolio et al)

which is found in the subset of atypical spitzoid melanomasor after specific carcinogenic treatments [79] thereby sup-porting the notion that CDK4 is important for melanomadevelopment

212 IntermediateLow Penetrance Genes Minor familialmelanoma loci have been more commonly found in thegeneral population with a lower penetrance of their germlinemutation than CDKN2A and CDK4

MITF MITF belongs to the MYC supergene family ofbasic helix loop helix transcription factors MITF is criti-cal for melanocyte cell-fate determination during commit-ment from pluripotent neural crest stem cells and is alsorequired for postnatal melanocyte functioning [80] WhileMITF has only been considered for many years as themaster regulator of melanocyte differentiation more recentdata have implicated MITF in the control of proliferationsurvival and the pathogeny of melanoma [81] Genomicamplification of MITF is found in 10 of primary and 20of metastatic melanomas and correlates with decreased 5-year overall patient survival yet it does not translate intoa strong increased expression at the protein level [82 83]The paradoxal function of MITF could be explained by avariation in its level of expression its different cofactors andits posttranslational modifications [84] It has been proposedthatMITF acts as a rheostat in themelanocyte lineage In thismodel a transient decrease in MITF expression is associatedwith a melanoma-initiating cell phenotype [85ndash87] whereasmoderate MITF level is linked with proliferation [84 88] andhigh MITF level with differentiation [14 18 19] Moreover

a sustain inhibition of MITF expression is associated with asenescence phenotype [30 89ndash91]

Posttranslational modifications of MITF also contributesto MITF activity Therefore MITF can be phosphorylatedand degradated via the ubiquitin-proteasome pathway inresponse to activation of the ERK pathway [92 93] Bysequencing the entire coding sequence of MITF in a highlyselected set of patients presenting either a strong fam-ily history of CMM or multiple primary melanomas ourgroup identified a germline missense substitution pE318K(c952GgtA NM 0002483) occurring at a significantlyhigher frequency in the at-risk patients than in the controlpopulation and conferring a 5-fold increased melanoma risk[39]Themutation was also reported in sporadic and familialcases in Australian and English cohorts and associated with a2-fold increasedmelanoma risk [42] Subsequently theMITFE318K variant was found in a group of Italian melanomapatients [40] and in another Australian study [41] withsimilar allele frequency All these studies show that theMITFE318K variant is enriched in those with multiple primarymelanomas

It was shown that the nonsynonymous c952GgtA substi-tution that changes the glutamate 318 into lysine replaces theSUMOylation consensus binding site IKQE in the C-terminalpart of MITF by the IKQK sequence and reduces MITFsumoylation [39] SUMOylation is an ubiquitination-likeposttranslational modification triggering covalent SUMOattachment to target proteins [94] which deregulation hasbeen involved in several human diseases

Expression of MITF E318K enhances the migrative andinvasive properties of melanoma cells and increases the

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 7

ability to form colonies of immortalized melanocytes henceindicating that MITF E318K displays protumoral properties[39]

According to its level and the type of posttranslationalmodification the repertoire of MITF target genes differsMITF has been reported to control expression of genesinvolved in cell proliferation (CDK2) [95] in cytoskeletonremodeling and migration [84] and in cell survival (BCL2BCL2A1 ML-IAP MET APE1 and HIF1a) providing antiox-idant defense and elevating the antiapoptotic features ofmelanocyte cells [27 28 96ndash98] Consistently MITF activitycorrelates directly with resistance to UV-induced apoptosisin melanocytes [99] MITF is also the major transcriptionalregulator of TRPM1 a transient receptor potential cationchannel which expression has been correlated with a highermetastatic risk in skin melanoma [100] Recently MITF wasreported to regulate the expression of PGC1120572 [101 102]a regulator of mitochondrial biogenesis and respirationgluconeogenesis as well as many other metabolic processes[103] More broadly MITF occupies a prominent positionin the melanocyte lineage working as a molecular hub thatdetermines the melanocyte behavior

MC1R The melanocortin 1 receptor (MC1R) is a seven-pass transmembrane G-protein coupled receptors expressedon the cell surface of epidermal melanocytes Upon stim-ulation by the proopiomelanocortin-derived peptides 120572-melanocyte-stimulating hormone (120572-MSH) and adrenocor-ticotropic hormone MC1R activates adenylate cyclase thecAMPPKACREB cascade and the pigmentation pheno-type Loss-of-function for MC1R is responsible of the so-called red hair color phenotype in individuals of northernEuropean ancestry Additionally genetic variation at MC1Rlocus is also an important risk factor for melanoma [104105] Therefore carriers of MC1R variants that correspondto the red hair fair skin phenotype (V60L R151C R160Wand D294H) have a 2ndash4-fold increased risk of developingmelanoma [106ndash108] Moreover the presence of an MC1Rvariant in addition to a CDKN2A mutation significantlyincreases the melanoma penetrance decreasing the age atonset compared with individuals carrying a CDKN2A muta-tion alone [109 110] Finally patients withMC1R variants hada 5- to 15-fold increased risk of BRAF-mutant melanomasregardless of signs of chronic solar damage [111 112]

Other Low-Risk Alleles Recent studies have focused ondifferent variants responsible for differences in pigmenta-tion of hair eyes and skin but also skin sensitivity toUV It has been shown that variants of pigmentation locisuch as in ASIP an MC1R receptor antagonist compet-ing with the 120572-MSHMC1R cascade and thus inhibitingthe constitutive pigmentation the melanogenic enzymesTYR and TYRP1 [113] or MATPSLC45A2 (membrane-associated transporter proteinsolute carrier family 45 mem-ber 2) involved in intracellular processing and traffickingof melanosomal proteins [114] are significantly associatedwith increased melanoma risk [113] In addition genes notinvolved in pigmentation were associated with an elevatedrisk of developing melanoma including germline mutations

in MTAP (methylthioadenosine phosphorylase) an enzymeplaying a major role in polyamine metabolism [115] andPLA2G6 encoding a phospholipase A2 group VI [115] TheTERT locus (at 5p1533) was found associated withmelanomarisk Recently a melanoma-segregating germline mutation inthe promoter of the telomerase reverse transcriptase (TERT)gene has been shown to create a new binding motif forEtsTCF transcription factors such as ELK1 and ELK4 nearthe transcription start In reporter gene assays this variantcaused up to 2-fold increase in transcription [116]

22 Acquired Genetic and Epigenetic Components Genescan also be targeted for mutations or deletions not inthe germline but as acquired events in individuals withsporadic melanomas Melanoma is a heterogeneous tumorand several molecular events revealed by genomic pro-teomics and candidate gene approaches have been identifiedand associated with its development In addition to thecommonly mutated genes BRAF NRAS PTEN TP53 andp16 new candidate genes have been identified GRIN2Awhich encodes a subunit of the glutamate receptor [117]ERBB4 a growth factor transmembrane receptor [118] andthe metalloprotease MMP8 [119] are mutated in 30 19and 7 of melanoma cases respectively More recently deep-sequencing approaches of melanoma samples of differentmelanoma types highlighted new melanoma driver genessuch as PREX2 PPP6C and RAC1 [55 56] Other mutationswere also reported such as in SNX31 that encodes the proteinsorting nexin 31 likely acting as a Ras effector protein [120]in TACC1 that stimulates the Ras and PI3K pathways andpromotes transformation in vivo [121] and in STK19 a kinasewith yet unknown function Functional studies are necessaryto clearly identify the role of some of these recent mutationsin melanoma pathogeny

Rather than establishing a catalog of all the molecularchanges reported so far in melanoma tumors this reviewwill be restricted to the main players of melanomagenesisIn addition to environmental factors cells must acquiresuccessive genetic lesions prior to forming tumors Thesealterations are discussed using a conductive wire based on theknown biological role of these players inmelanoma initiationand progression

In aggregates a high frequency of activating BRAFmutations (80) was identified in nevi indicating thatactivating BRAF mutations occurs early during melanomaprogression [122] an idea supported by recent findingsshowing that the 119861119877119860119865V600E mutation was present in themajority of if not all of melanocytes in the 119861119877119860119865V600Enevi examined [123] Activating BRAF mutations triggeredan initial burst of cell proliferation followed by inductionof senescence The primary mediator of senescence in neviappeared to be p16INK4a blocking cyclin D1CDK4 complexesand preventing cell proliferation KIT alsomediated cell cyclestimulating activities yet its effect seemed to be restricted toa subset of melanomas PTEN loss triggered activation ofthe PI3KAKT pathway and escaped of BRAFV600E-mediatedsenescenceTherefore PTEN loss could terminate the bypassof senescence initiated by p16INK4a loss and via PI3KAKT

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Scientifica

pathway could favor melanoma progression Deregulationof the PI3KAKT pathway mostly occurred as a late-stageevent in melanoma implying that it operated in malignantprogression more than in melanoma initiation This notionwas supported by a study in which the functional impactof the RASBRAF and PI3KAKT signaling pathways wasinvestigated using reconstructed skin [124] Expression of thecatalytic subunit of PI3K produced invasivemelanocytic neo-plasia while only mild junctional hyperplasia was seen uponBRAFV600E expression RAC1 one of the key targeteffectorof PI3K regulates cell motility The metastasis process isalso associated with acquisition of mesenchymal propertiesa process assimilated to an EMT program that is favored bythe hypoxic environment of the skin [125] (Figure 3)

221 The RASRAFMEKERK Cascade Ras family consistsof three isoforms HRAS NRAS and KRAS each encod-ing a membrane-localized small GTPase The members arecomposed of a catalytic domain that mediates the guaninenucleotide binding and hydrolysis and of an hypervariableregion containing the membrane targeting domain requiredfor its activation They function as a molecular switchlinking receptor and nonreceptor tyrosine kinase activationto downstream cytoplasmic or nuclear events The familyof serinethreonine kinases RAF has three isoforms ARAFBRAF and CRAF (RAF-1) activated by the small GTPasesRAS

Oncogenic RAS or BRAF alone appears relatively poorin inducing melanoma transformation unless they are com-bined with other genetic alterations In human 81 ofcongenital melanocytic nevi harbor RASQ61KR mutationsand 82 of acquired nevi harbor BRAFV600E mutation[126] Forced expression of oncogenic NRAS or BRAF innormal melanocytes triggers a senescence phenotype [127128] a notion that supports the idea that nevi have agrowth arrest via oncogene-induced senescence Nevi canremain arrested in growth for decades Mouse modelsexpressing NRASQ61K [68] or BRAFV600E [129 130] developbenign melanocytic lesions characteristic of nevi that rarelyprogress to melanoma strengthening the previous notionHowever melanoma formation in mouse model is greatlyaccelerated in the absence of p16INK4a [68] Pten [129] or120573-catenin [131] In human also it is thought that severalgeneticepigenetic alterations andor additional environmen-tal stimuli are needed to drive melanoma development [132]

Almost 80 of melanomas have either BRAF or NRASmutations [133] Most common melanoma mutations are inNRAS (15ndash30 of the cases) among which themost frequentare substitutions of glutamine at position 61 by a lysine oran arginine (Q61K Q61R) [134] However HRAS mutationcan also be found in the subset of sptizoıd melanomas [135]In 2002 oncogenic mutations in the serinethreonine kinaseBRAF was identified in nearly 70 of cutaneous melanomas[136] a percentage closer to 50 when considering a highernumber of melanoma specimens [33 137] The T1796rarrAtransition leading to the V600E change at exon 15 representsnearly 90 of the casesThismutation creates a constitutivelyactive status for BRAF independent of a previous activation

by RAS oncogene and extracellular stimulus BRAF muta-tions are associated with high level of UVR in early lifecompared to patients with NRAS mutation who have hightotal exposure spread throughout life Patients with BRAFmutations are younger and have greater number of neviNRAS and BRAF mutations are mutually exclusive yet veryrare exceptions were reported [138]

Oncogenic BRAF transforms immortalized melanocytes[139] and it stimulates proliferation of melanoma cellsSimilarly introduction of MEK into murine immortalizedmelanocytes leads to tumorigenesis in nude mice [140]Conversely inhibition of Ras [141] BRAF [142ndash144] or MEKblocks ERK activity and inhibits the growth of melanomacells both in vitro and in vivo

Recently NRASBRAF activation was shown to mediatean epithelial-to-mesenchymal transition (EMT) switch inlate-stage melanoma that relies on TWIST1 ZEB1 and E-cadherin loss and results in enhanced invasion This EMTprogram constitutes an independent factor of poor prognosisin melanoma patients [145]

This NRASBRAF signaling pathway has attracted con-siderable attention as a target for anticancer therapy becauseof its high frequency of mutations and its important role inmelanoma disease The novel cancer therapeutic approachesbased on the inhibition of some members of this cascade willbe developed and discussed later in Section 3

222 CCND1 The CCND1 gene encodes cyclin D1 anactivating subunit of the CDK4 and CDK6 kinases whichcontrol the G0G1 cell cycle progression Cyclin D1 is aprotooncogene playing an important role in cancer includingmelanoma Gene amplification of chromosome 11p13 con-taining the CCND1 locus and enhancing cyclin D1 expres-sion has been observed in melanoma cell and particularlyin acral (444) lentigo malignant (105) and sinonasalmelanomas (625) [146 147] Cyclin D1 overexpressionmight increase resistance to BRAF inhibitors [148] althoughthis observation was not confirmed in further studies [149]

Cyclin D1 silencing triggers a G1S cell cycle arrest in vitro[150] and leads to inhibition of melanoma growth in vivo[147]

223 KIT KIT is a type III transmembrane receptor tyro-sine kinase Binding of its ligand stem cell factor (SCF)results in receptor dimerization autophosphorylation andactivation of several signaling pathways thereby mediat-ing cancer cell growth proliferation invasion metastasisand inhibition of apoptosis The SCFKIT pair plays a keyrole in melanocyte specification during embryogenesis andmelanocytes differentiation and proliferation [151ndash153] KITis expressed in more than one-half of early-stage malignantmelanomas [154] However loss of KIT expression has beenobserved with progression of disease from superficial andinvasive to metastatic stages suggesting that KIT possessestumor suppressive functions [155] Loss of KIT expressionis thought to be mediated through AP-2120572 [156] The AP2transcription factor family is a set of retinoic acid induciblegenes regulated along the development and composed of four

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 9

p16INK4Alowast

(CDK4lowast activation)

p14ARFlowast (TP53 loss)

Melanocytenevidysplasic nevi

Epid

erm

isD

erm

is

Vertical growthphase Metastatic phaseRadial growth

phase

Basal membrane and stroma degradationneoangiogenesis metastasis

Dysregulation of growthapoptotic pathways EMT

Loss of keratinocyte control immune system escape

NRASQ61KR

BRAFV600EPTEN E-cadherin

MMP29 WNT5ALossLoss TRPM1Gain BRN2

MITFlowast KIT Pten TP53

Immortalization InvasionProliferation Metastasis

120573-catenin CCND1 TERTlowast

Figure 3 Hypothetical model of Melanoma development In 25 of cases melanoma derives from a pre-existing nevus through a multistepprocess regulated by a key set of genes Cellsmust acquire successive genetic lesions prior to forming tumors andmetastases Asterisks indicategenes mutated in the germline

related factors AP2120572 AP2120573 AP2120574 and AP2120575 AP2 factorsorchestrate a variety of cell processes including apoptosiscell growth and tissue differentiation during embryogenesisAP2120572 has been shown to function as a tumor suppressorby regulating the transcription and expression of p53 [157]A dominant-negative AP2120572 mutant enhances invasive andtumorigenic properties of melanoma cells [156] KIT isactivated by mutation in only 2ndash6 of cutaneous melanomasbut aberrations in KIT (mutation amplification) have beenreported in melanomas of acral (36) mucosal (39) andchronic sun-damagedlentiginous (28) types [158ndash161] Itis interesting to note that in the majority of melanomaswith mutations in BRAF or NRAS the expression of c-KITseems reduced to allow tumor progression [162] Howeveran overexpression of KIT and CDK4 has been identified ina subgroup of cutaneous malignant melanomas and wouldbe a mechanism of potential oncogenic transformation ofnonmutated BRAF or NRAS melanomas [163]

224 The Tumor Suppressor Gene TP53 The TP53 geneencodes the transcription factor p53 activated in responseto various stresses including DNA damage hypoxia orexpression of aberrant oncogene p53 regulates positivelyor negatively many genes involved in cell cycle regulation(CDKN1A) induction of autophagy senescence and apop-tosis (NOXA PUMA and BAX) as well as genes involved inthe DNA repair or cellular metabolism [164]

The importance of p53 in maintaining cellular integrityis underscored by the fact the TP53 knockout mice spon-taneously develop tumors [165] Mutations or deletions of

TP53 are found in approximately 50 of human cancers yetin melanoma the ratio is lower 1ndash5 primary melanomasand 11ndash25 metastatic melanoma harbor mutated p53 [166]Most of the melanoma cases harboring p53 mutation (19in the discovery set) were without concurrent mutation inCDKN2A locus [56] Why frequency of p53 mutation is low-est in melanomas compared to other cancers remains to besolved In human melanoma cells deregulation in upstreamregulators or downstream effectors indicate that the p53signaling cascade is nevertheless compromised Moreovercompelling evidence supports a role for p53 in melanoma-genesis Mouse and zebrafish models harboring oncogenicBRaf or Ras develop benign melanocytic hyperplasias thatresemble nevi while melanomas occur in a p53-deficientbackground thereby indicating that p53 restrains tumorprogression [167ndash170] Therefore melanoma cells show adecreased expression level in the proapoptotic p53 effectorAPAF1 following methylation of its promoter [171 172]Additionally MDM2 the E3 ubiquitin ligase responsiblefor p53 ubiquitylation and degradation via the proteasomeis frequently overexpressed in melanoma cell lines [173]Recently MDM4 over MDM2 has been shown to be overex-pressed in cells freshly isolated from primary and metastaticmelanomas compared to cultured melanoma cell lines thelater being likely selected for MDM2 overexpression [174]These observations could explain why nutlin-3 an inhibitorof the MDM2-p53 interaction provides poor clinical benefitStrategies to impair the MDM4-p53 interaction are currentlyunder development Stapled peptides that bind with high

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Scientifica

affinity to MDM4 already provide promising antimelanomaeffect both in vitro and in animal model experiments [174]

p53 negatively regulates hypoxia-inducible factor 1120572(HIF-1120572) a factor that facilitates melanoma invasion anddevelopment of more aggressive tumors [175 176]

p53 also has a role in regulating both EMT and EMT-associated stem cell properties p53 suppresses EMT byrepressing expression of ZEB1 ZEB2 and SNAIL throughregulation of microRNA including members of the miR-200family and miR34 [177 178]

225 The PI3KAKT Signaling Pathway The PI3KAKT cas-cade can be activated by mitogenic stimuli or growth factorssuch as IGF1 or by RAS PI3K catalyzes the phosphoryla-tion of phosphatidylinositol (PI) into phosphatidylinositol-3 phosphate (PIP3) which recruits PDK1 and triggers theactivation of the serinethreonine kinase AKT Phosphataseand tensin homologue deleted on chromosome 10 (PTEN)is a phosphatidylinositol phosphate phosphatase and is fre-quently inactivated in human cancers There are three formsof AKT AKT1 AKT2 and AKT3 In melanoma cells AKT3is the form preferentially expressed AKT3 activation isfound in about 60 of sporadic melanomas subsequent togene amplification (35 of the cases) or to inactivation ofPTEN (40ndash60 of the cases) which negatively regulates thePI3KAKT pathway [179ndash181] Mutations of PI3K have beenidentified in only 5 of the cases [181] Mutations in thePI3K signaling cascade were also discovered inMTOR IRS4PIK3R1 PIK3R4 PIK3R5 and NFKB1 [182]

Interestingly while PTEN loss is found at high frequencyin melanoma (37) but not in nevi [181] AKT3 activity levelincreases with advancing melanoma stage [181] These obser-vations support the notion that alteration of the PI3KAKTcascade is not an early event in melanoma progression

NRAS and PTEN mutations were first reported to bemutually exclusive in melanoma which is likely explainedby their ability to share the same signaling pathway [183]However recent data indicate that PI3K pathway mutationscooccurred with 9 of NRASmutant tumors [182] Cooccur-rence of BRAF and PTEN mutations was reported in 17 ofmelanomas [182]

Inhibition of AKT3 promotes an inhibition of tumorgrowth in vivo and is associated with an increase in apoptosisin vitro [181] Reintroduction of PTEN reduces growth ofmelanoma xenografts [180]

More broadly the PI3KAKT signaling pathway con-tributes to the phenotypic plasticity of cancer cells by control-ling EMT [184]This pathway is also embroiled in invasion byregulating expression and activity of factors such as RAC1involved in cell motility and in degradation of basal laminaecomponents such as the metalloproteases MMP-9 [185]allowing melanoma cells to invade the underlying dermis[186]

226 The Epithelial-Mesenchymal Transition Epithelial-to-mesenchymal transition is the process in which epithelialcells lose their epithelial characteristics gain mesenchymalfeatures and become motile Cells that undergo EMT could

also gain stem cell-like properties [187] EMT orchestratesthe lost of the cell-cell and cell-extracellular matrix (ECM)interactions and confers the ability to migrate throughinduction of a new genetic program specifically increasedexpression of SNAILSNAI1 SLUGSNAI2 ZEB1 ZEB2SIP1TWIST proteins and E47 and decreased expression of theadherence molecule E-cadherin [188ndash190]

EMT plays a crucial role during different stages ofembryonic development [191] and can be reactivated in anadult organism under some circumstances such as tumorige-nesis EMT-promoting pathways are associated with differentsignaling molecules such as transforming growth factorbeta (TGF-120573) epidermal growth factor (EGF) fibroblastgrowth factor (FGF) hepatocyte growth factor (HGF) bonemorphogenetic proteins (BMPs) and WNTs and NotchTherefore EMT programs emerge as important regulators ofphenotypic plasticity in cancer cells

EMT has been shown to play an important role in medi-ating the invasion and metastasis of epithelial tumors as wellas melanoma cells [192] In order to invade the underlyingepidermismelanoma cellswill downregulate somemoleculesinvolved in cell-cell adherent junctions such as E-cadherinleading to their detachment from the basal laminae and toadjacent keratinocytes Melanomas do not show a classicalEMTand they adoptmoremesenchymal features E-cadherinexpression may be repressed at the transcriptional level bySlug Snail or Twist [193ndash195] In this regard SLUG mightfacilitate the invasive properties of melanocytic cells [196]In the context of melanoma experimental regulation of E-cadherin expression controls cancer progression in somemouse models [197] E-cadherin reintroduction decreasescell growth survival and invasion [198] Interestingly thelevel of E-cadherin correlates with MITF expression [199]

EMT is also regulated by the PI3KAKT pathway throughseveral routes AKT enhances Mdm2-mediated ubiquitina-tion and degradation of p53 [200] and p53 regulates theepithelial-mesenchymal transition [201] Wnt signaling alsoregulates GSK-3120573 and thereby supports EMT by downstreameffects on SNAIL and SLUG [202] Additionally AKT anda downstream effector RAC1 increase Snail expression [203204]

These observations support the role of the PI3KAKTpathway in conferringmelanoma cells invasive and stem cell-like features

227 TGF120573 TGF-120573 signaling regulates melanoma tumori-genesis and metastasis [205] and is a major inducer of EMT[206] The members of the SMAD family are activated bybinding of TGF-120573 ligands to their cellular receptors then theyaccumulate in the nucleus where they control the transcrip-tion of target genes GLI2 a critical Sonic hedgehogmediatorhas been identified as a direct transcriptional target of theTGF-120573SMAD pathway in melanoma cells [207] High GLI2expression is associated with a more aggressive phenotypecharacterized by loss of the cell-cell adhesion molecule E-cadherin hallmark of cancer progression [208]

Increased expression and secretion of TGF-1205732 and TGF-1205733 seem to occur early in melanoma progression and to

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 11

increase with tumor progression A correlation betweenTGF-1205732 expression and tumor thickness has been reportedand TGF-120573 is associated with an invasive signature [209]

228 The Wnt120573-Catenin Signaling Pathway WNT aresecreted glycoproteins involved in developmental processesand in the maintenance of cellular homeostasis such as cellproliferation cell polarity stem cell self-renewal and cell-fatedetermination [210] Particularly theWNT signaling cascadeplays a crucial role in the neural crest induction specificationand melanocyte differentiation

The Wnt signaling might reflect the combination of atleast three different signaling branches the Wnt120573-catenincanonical branch controls cell proliferation and differenti-ation and the noncanonical branch involving the WntCa2+and planar cell polarity (PCP) pathways is involved incytoskeleton organization and cell motility

Wnt1 and Wnt3a are considered as the canonical ligandsand Wnt5a as an activator of the WntCa2+ pathway

In the canonical pathway binding of the Wnt proteins totheir receptor Frizzled (Fz) and coreceptor of the LRP family(only LRP56 are expressed in vertebrates) increases the poolof cytoplasmic 120573-catenin by preventing its phosphorylationand degradation through a complex including the tumorsuppressor Adenomatous Polyposis Coli (APC) the scaffoldprotein Axin2 casein kinase 1 (CK1) and Glycogen SynthaseKinase 3 (GSK3) This leads to 120573-catenin translocation to thenucleus where it transactivates transcription factors of theTCFLEF family and controls the transcription of target genessuch as cyclin D1 c-MYC MITF and BRN2 and stimulatescell growth [7 211ndash213] TGF-120573 is known to activate thecanonical Wnt pathway This pathway is activated in morethan 30 of melanomas as illustrated by the presence of120573-catenin in their nucleus [214] with approximately 3harboring 120573-catenin mutations [215]

In mice a form of 120573-catenin constitutively localized atthe nucleus is not sufficient by itself to induce the forma-tion of melanoma but causes melanocyte immortalization[216] To do so 120573-catenin seems to repress the promoteractivity of p16INK4a which controls the senescence programand prevents cellular immortalization However an activeform of 120573-catenin increases the penetrance and incidenceof melanoma in mice expressing oncogenic NRASQ61K orBRAFV600E specifically in melanocytes [131 216]

Additionally expression of negative regulators of canon-ical Wnt signaling pathway such as Dickkopf-1 2 3 (Dkk-12 3) and Wnt inhibitory factor-1 (WIF-1) is strongly reducedor lost both in melanoma cell lines and tumor samples [217218] Forced expression of DKK1 orWIF-1 reducesmelanomacell growth and activates cell death [219 220]

However the role of the Wnt120573 catenin pathway inmelanomagenesis is complex and contradictory data havebeen reported Metastatic progression is associated with theloss of nuclear 120573-catenin and the accumulation of 120573-cateninin cell nuclei of both primary tumors and metastases is amarker for good prognostic for patients [221]

Additionally B16 melanoma cells expressing WNT3Aimplanted into mice exhibit decreased tumor size and

decreased metastasis Moreover WNT3A upregulates genesinvolved in melanocyte differentiation several of them aredownregulated with melanoma progression [221] Consis-tently meta-analysis of melanoma gene expression revealedthat the Wnt pathway is associated with high proliferationand low metastatic features [222] Furthermore BRAF sig-naling activated in most of melanomas through mutationor autocrineparacrine activation inhibits Wnt120573-cateninsignaling in humanmelanoma cells and 120573-catenin is requiredfor PLX4720-induced apoptosis in melanoma cells [223]

Collectively these discrepancies could be explained bythe type of Wnt ligands and signaling cascades initiated bythe level of nuclear 120573-catenin and the tumor stage

Nevertheless elevated expression of WNT5A is fre-quently associated with high grade melanomas [224 225]WNT5A forced expression in low metastatic melanomaincreases their aggressive features [225]

229 RAC1 TheRho family genes include more than twentymembers and encode GTP hydrolases The most extensivelycharacterized members are RAC1 and RHOA [226] TheseGTPases coordinate various cellular functions includingcell polarity motility vesicular trafficking cell cycle andtranscriptomal dynamics [227 228] RAC1 interacts with p21-activated protein kinase 1 (PAK1) to regulate downstreamevents essential in tumorigenesis Recently a mutation inRAC1 has been discovered in melanoma and ranked asthe third most frequent hot spot gain-of-function mutationoccurring in melanoma after those in BRAF and NRASThis RAC1-P29S substitution releases the conformationalrestraint conferred by the conserved proline which inducesan increased binding of the protein to downstream effectorsand promotes melanocyte proliferation and migration [57229]

Additionally aberrant activation of upstream regulatorsof RAC1 particularly in theDBL family of guanine nucleotideexchange factors (GEF) specific for RAC1 (eg TIAM1PREX1-2 and ECT2) has been implicated in various cancersKnock-out of PREX1 upregulated during melanomagenesisreduces melanoma metastasis [230] Recently mutations inanother member of the P-REX family PREX2 have beenreported yet their role in melanoma disease remains to beclarified [55]

Rac1 inhibition using pharmacological or geneticapproaches was reported to impair melanoma tumor growthand spreading to distant organs in the TyrNRas (Q61K)mouse model suggesting a potential value for RAC1 as atherapeutic target at least in some context [231]

3 Current and Emerging Approaches inMelanoma Treatment

As mentioned above early stage melanoma localized to theskin can be cured by surgical excision But most patients withunresectable stage III or stage IV disease require systemictreatment

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

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BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Scientifica

31 Conventional Chemotherapies Conventional chemother-apy is based on the use of alkylating agents such as fotemus-tine (Muphoran) dacarbazine (Deticene) and temozolomide(Temodal) which trigger cytotoxic effects by blocking cellreplication However these chemotherapy drugs promoteonly 10 of objective response with no improvement of over-all survival [179] Since the major breakthrough realized in2011 with the FDA approval of vemurafenib (V600E mutatedBRAF inhibition) for mutated BRAFV600E melanomas thesedrugs are limited to patients harboring non-BRAFV600Emutatedmelanomas or for patients who developed resistanceto previous treatments

32 Personalized Therapy The recent characterization of themolecular alterations in melanoma leads to the developmentof targeted therapies These treatments are designed to targettumors according to their molecular diversity and activatedintracellular signaling pathways

321 Targeting the BRAFMEKERK Signaling Pathway Thediscovery that BRAF is activated by mutation in a highpercentage of melanoma specimens opened the door to thesearch for BRAF inhibitorThe first agent developed to targetoncogenic BRAF was sorafenib (BAY 43-9006 Nexavar)a multikinase inhibitor that inhibited BRAF (wild-type orV600E) but also VEGFR PDGFR cKIT and FLT3 Thisdrug proved to be inefficient in the treatment of unresectableor metastatic melanoma [232] Significant efforts have beenspent to develop more specific and effective BRAF inhibitorsleading to the discovery of vemurafenib (previously knownas PLX-4032 currently marketed as Zelboraf) the first drugtargeting mutated BRAFV600E while having nofew effect onwild-type BRAF In phases 1 and 2 clinical trials vemurafenibshowed an objective response rate gt50 in patients sufferingfrom melanoma The results were confirmed in a phase 3clinical trial (BRIM3) where unpreviously treated patients(119899 = 675) showed response rates of 48 for vemurafenibversus 5 for dacarbazine a PFS of 53 months for vemu-rafenib versus 16 months for dacarbazine and at six monthsthe overall survival was 84 in the vemurafenib group and64 in the dacarbazine group [233] With a median follow-up of 125 months for patients treated with vemurafeniband 95 months for those initially receiving dacarbazinethe PFS was significantly improved with vemurafenib (69versus 16 months for dacarbazine) the overall survival wassignificantly prolonged with vemurafenib (136 versus 97months) and the objective response rate was significantlyhigher with vemurafenib (57 versus 86)

Vemurafenib received the FDA approval in 2011 How-ever a subset of BRAFV600E patients were initially resistantto vemurafenib and most of the others developed secondaryresistance Nearly all tumors demonstrated reactivation of theMAP kinase pathway with elevation of ERK phosphorylationat the time of resistance Different mechanisms involvedin acquired and secondary resistance have been reported[234] Moreover a metabolic rewiring linked to oxidativephosphorylation and controls by the MITFPGC1120572 axis hasbeen involved in vemurafenib resistance

Furthermore vemurafenib showed adverse effects suchas the development of cutaneous squamous cell carcinomas(SCCs) [235] through paradoxical activation of MAPKsignaling (about 20ndash25 of the patients with advancedmelanoma) [236]

Therefore there is an urgent need to subvert an eventualdrug resistance andor further improve the clinical outcomeof the patients

In this context other BRAF inhibitors are under devel-opment Dabrafenib (marketed as Tafinlar) has received anFDA approval for unresectable or metastatic melanoma withBRAF mutations in 2013 Dabrafenib is in the same class asvemurafenib working with a similar efficiency but seemsto be more efficient in melanoma brain metastasis [237]In the pivotal phase 3 clinical trial (119899 = 250) dabrafenibsignificantly increased the response rate compared to dacar-bazine (nearly 50 versus 6) and PFS (median 51 months)compared with dacarbazine (median 27months) Cutaneousside effects are also common with dabrafenib

Therapeutic combinations are actually launched toreplace single-agent BRAF inhibitors Combination of aBRAF inhibitor and a MEK inhibitor exhibited reducedincidence of skin toxicity including the development of skincancers presumably because the MEK inhibitor blocks thisparadoxical activation of the MAPK pathway

Trametinib and MEK162 are potent highly specificinhibitors of MEK1MEK2 BRAF downstream kinases [238ndash240] giving responses in 20 of the melanomas harboring aBRAF mutation

Trametinib (marketed as Mekinist) has also been FDAapproved for unresectable or metastatic melanoma withBRAF V600E or V600K [239] Its efficiency was demon-strated in the phase 3 Metric trial (119899 = 322) where PFS fortrametinib was improved compared with either dacarbazineor paclitaxel (median 48 versus 15 months) as well as overallsurvival (81 versus 67 resp at 6 months)

322 Targeting KIT KIT is mutated in 15ndash20 of patientswith acral or mucosal melanomas and withmelanoma occur-ring in areas of chronic skin damage Imatinib mesylatea kinase inhibitor targeting bcr-Abl c-kit platelet-derivedgrowth factor receptor (PDGFR)-alpha and PDGFR-beta andinducing remarkable clinical responses in several cancerswas therefore used for melanoma Although many activat-ing c-KIT mutations have been reported c-KIT mutantmelanoma appears initially sensitive to imatinib [241] It hasbeen demonstrated in vitro that the tyrosine kinase inhibitorimatinib mesylate (Glivec) inhibits proliferation and inducesapoptosis in melanoma cells with hyperactivation of c-KITThese biological effects go through an increase in p27KIPand inhibition of the ERK PI3KAKT and STAT signalingpathways [163 242] However Todd et al have showed thatthe majority of patients treated with this inhibitor wouldeventually progress Secondary mutations in c-KIT werereported to mediate this resistance Cells with an A829P c-Kit mutation are resistant to imatinib but are still sensitiveto the tyrosine kinase inhibitors nilotinib and dasatinibAdditionally the T670I c-Kit mutation mediates resistance

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 13

to imatinib nilotinib and dasatinib but remains sensitive tosunitinib [241]

Phase II studies using imatinib in unselected groupsof patients with advanced melanoma demonstrated onlyminimal evidence of activity [243 244] However phase IIclinical trials conducted on patients with c-KIT mutationsshowed objective response rates in 33 of cases [245 246]Moreover results from a phase II trial indicated that imatinibcould be effective when tumors harbored KIT mutations butnot if KIT only was amplified [247]

33 Immunotherapy It has been well documented thatmelanoma was an immunogenic tumor but metastaticmelanoma cells have developed mechanisms to escape fromimmunosurveillance and to survive The immune systeminvolvement in protection against melanoma is supportedby the increased melanoma incidence under immunosup-pression conditions Conversely some melanomas whichshowed spontaneous regression and spontaneous infiltrationof the tumor by T-lymphocytes seemed to be a factor ofgood prognosis [248] These data led to the conclusion thatimmunological strategies could improve the prognosis ofmetastatic melanoma [249] In 1998 the FDA approved theuse of the immune molecule interleukin-2 (IL-2) to treatadvancedmelanomaThe immunemolecule interferon alpha(IFN120572) has also been used alone after surgery or in combina-tion with other agents to treat advancedmelanoma Howevermost of clinical trials based on immune system activationdid not translate into clinicallymeaningful objective responserate and any improvement in overall survival

Therefore development of novel therapeutic approachesalong with optimization of existing therapies continues tohold a great promise in the field of melanoma therapyresearch Promise for the use of immunotherapy in thetreatment of melanoma has been highlighted recently

331 CTLA4 Blocking Antibody In tumors ectopic cytotoxicT lymphocyte-associated antigen 4 (CTLA-4) bounds to itsligands B71 and B72 on T-cells and caused inhibition of T-cells activity [250] Following T-cell activation CTLA-4 wasrecruited to the plasma membrane where it functioned inan autoregulatory role attenuating T-cell activation and pro-liferation thereby restraining effective antitumor immunityThese observations suggested the potential value of an anti-CTLA4 approach in the treatment of metastatic melanomaIpilimumab (Yervoy) a novel antibody blocking CTLA-4induced an overall survival benefit in two randomized phaseIII studies (119899 = 676 and 119899 = 502 resp) [251 252] and receivedan FDA approval in 2011

The median overall survival was 10ndash112 months amongpatients receiving ipilimumab plus gp100 or ipilimumab plusdacarbazine as compared with 64 months among patientsreceiving gp100 alone [251] and 91 months among thosereceiving dacarbazine [252]

The median overall survival with ipilimumab alone was101months [251]Major drawbacks of this treatment were thelow rate of objective response (10) a minority of patients

achieving long-term disease control and the serious sideeffects

332 PD1 Blocking Antibody The programmed death 1 (PD-1) receptor is an inhibitory receptor expressed at the surface ofactivated T cells When PD-1 attaches to programmed deathligand-1 (PDL-1) expressed on cancer cells the T cellrsquos abilityto target the tumor cell is inhibited

Recently monoclonal anti-PD-1 antibody lambrolizumab(MK-3475) was assessed in metastatic or unresectablemelanomas (119899 = 135) Objective response rate was obtainedin 38 of patients and the responses were sustainable in themajority of patients (median follow-up 11 months amongpatients who had a response) [253]

Additionally according to a phase 1 trial result includ-ing patients receiving a combination of nivolumab (BMS-936558) another PD-1 blocking antibody and ipilimumab(119899 = 53) versus patients who received a sequenced treatment(119899 = 33) the combo provided deep rapid and long lastingtumor responses in patients with advancedmelanoma 53ofpatients with a concurrent therapy demonstrated an objectiveresponse compared to the 20 objective-response rate of thegroup with sequenced treatment [254]

4 The Melanoma-Initiating Cells

Stem cells are undifferentiated cells characterized by theproperty of self-renewal They can multiply into identicalcells almost indefinitely or differentiate into specialized cellsMelanocyte stem cells (MSC) have been identified in thebulge of hair follicles [255] They self-renew to maintain thepool of MSC and differentiate into functional melanocyteswhose function is to color the growing hair Incomplete MSCmaintenance through SOX10 MITF or Bcl2 loss triggers hairgraying [255 256] A pool of MSC has also been identifiedfrom the dermis of glabrous skin by M Herlynrsquos group yettheir function remains to be elucidated [257]

Tumor cells with stemness features have also been char-acterised in several cancers including melanoma wheredifferentiated andmelanotic cells endowed with fast-growingproperties coexisting with cells growing slowly These slow-growing cells are thought to mediate chemoresistance andrelapses Therefore it appears of paramount importance todetermine their biological properties to develop efficientantimelanoma therapies Thus within a tumor tumor cellsproliferating rapidly but not individually competent to gen-erate a new tumor-initiating cell can be distinguished fromtumor cells proliferating very slowly but are able to generatea tumor The existence of melanoma-initiating cells has onlybeen recently proposed with the description of a subpopu-lation of melanoma cells expressing markers such as CD20[258] CD133 [259] CD24 [260] CD271 [261] or the ABCtransporter (ATP-binding cassette) involved in drug efflux(ABCB5) [262] ALDH1A [263] According to these studiesthe initiating cells which represent 16 to 20 of melanomacells were able to differentiate self-renew and establishclinically heterogeneous tumors in mice [258 259 262] Forexample the use of an antibody directed against ABCB5

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Scientifica

prevented tumor formation or decreased tumor growth of atumor already established [262]

Later on several groups have validated the presenceof melanoma-initiating cells identified as a minor subpop-ulation of cells with slow growing properties but with ahigh tumorigenic potential in vivo [85 264] These cellswere required for tumor maintenance and to form a het-erogeneous tumor when implanted into immunocompetentmice Inhibition of this cell subpopulation either by forcingtheir differentiation [85] or promoting a metabolic rewiring[265] decreased tumor formation Although the cellularheterogeneity found in the tumor might reflect the existenceof different cell types the studies also suggested the existenceof a phenotypic transition allowing the same cells to acquiredifferent phenotypes [87] Indeed JARID1B-negative cellscould become positive [264] and ABCB5+ melanoma cellsgenerate both ABCB5+ and ABCB5minus progeny [262] Thedirect implication of MITF in this phenotypic transition hasalso been demonstrated [85]

The existence of initiating cells in melanoma may explainthe lack of objective responses to cancer treatment and therisk of recurrence observed in most cases The standardtreatment formelanoma is based on chemotherapywith alky-lating agents (Decitene Temodal and Muphoran) targetingproliferative cells It is therefore not surprising that thesedrugs are poorly efficient on the slow-growing melanoma-initiating cells In addition melanoma cells are able toreduce the intracellular drug accumulation by increasing thelevel pumps efflux known as ABC transporters ABCB5presenting an increased expression during the malignanttransformation [266] has been involved in the resistance ofmelanoma cells to doxorubicin [267] Inhibition of ABCB5dramatically improved intracellular drug accumulation andreduced the resistance of melanoma cells to doxorubicin[267]

The melanoma-initiating cells are able to escape theimmune system These cells are able to mask their antigenicidentity inhibiting or not expressing such antigen MART-1(melanoma antigen reconnu by T cells-1) recognized by Tlymphocytes [268] or by inhibiting expression of the majorhistocompatibility complex type 1 (CMH-1) present antigenicpeptides to lymphocytes [268] For example ABCB5 positivecells show no MHC-1 or MART-1 Interestingly MITF reg-ulates the expression of these proteins [30 269] reinforcingour observations that cells expressing low levels of MITFcorrespond to tumor-initiating cells

During these last few years differences in the percentageof melanoma initiating cells (MIC) per tumor and the lackof consistent and reproducible markers of choice for MICisolation from one study to another have engendered astirring debate Therefore it is of paramount importance toidentify appropriate anduniversal surfacemarker(s) to isolateand characterize the MICs Biological characterization of thedifferent cells within a tumor will help researchers to puttogether a complete portrait of the tumors andwill contributeto design more efficient antimelanoma therapy [270]

References

[1] M Osawa G Egawa S-S Mak et al ldquoMolecular characteriza-tion of melanocyte stem cells in their nicherdquo Development vol132 no 24 pp 5589ndash5599 2005

[2] K Schouwey V Delmas L Larue et al ldquoNotch1 andNotch2 receptors influence progressive hair graying in a dose-dependent mannerrdquo Developmental Dynamics vol 236 no 1pp 282ndash289 2007

[3] Y Aoki N Saint-Germain M Gyda et al ldquoSox10 regulates thedevelopment of neural crest-derived melanocytes in XenopusrdquoDevelopmental Biology vol 259 no 1 pp 19ndash33 2003

[4] D Lang M M Lu L Huang et al ldquoPax3 functions at a nodalpoint in melanocyte stem cell differentiationrdquo Nature vol 433no 7028 pp 884ndash887 2005

[5] M Moriyama M Osawa S-S Mak et al ldquoNotch signalingvia Hes1 transcription factormaintains survival of melanoblastsand melanocyte stem cellsrdquo Journal of Cell Biology vol 173 no3 pp 333ndash339 2006

[6] S Shibahara K-I Yasumoto S Amae et al ldquoRegulation ofpigment cell-specific gene expression by MITFrdquo Pigment CellResearch vol 13 no 8 supplement pp 98ndash102 2000

[7] K Takeda K-I Yasumoto R Takada et al ldquoInduction ofmelanocyte-specific microphthalmia-associated transcriptionfactor by Wnt-3ardquo Journal of Biological Chemistry vol 275 no19 pp 14013ndash14016 2000

[8] S Agarwal and A Ojha ldquoPiebaldism a brief report and reviewof the literaturerdquo Indian Dermatology Online Journal vol 3 no2 pp 144ndash147 2012

[9] J Y Lin and D E Fisher ldquoMelanocyte biology and skinpigmentationrdquo Nature vol 445 no 7130 pp 843ndash850 2007

[10] S-I Nishikawa and M Osawa ldquoGenerating quiescent stemcellsrdquo Pigment Cell Research vol 20 no 4 pp 263ndash270 2007

[11] T Tumbar G Guasch V Greco et al ldquoDefining the epithelialstem cell Niche in skinrdquo Science vol 303 no 5656 pp 359ndash3632004

[12] R Cui H R Widlund E Feige et al ldquoCentral role of p53 in thesuntan response and pathologic hyperpigmentationrdquo Cell vol128 no 5 pp 853ndash864 2007

[13] Z Abdel-MalekMC Scott I Suzuki et al ldquoThemelanocortin-1 receptor is a key regulator of human cutaneous pigmentationrdquoPigment Cell Research vol 13 no 8 supplement pp 156ndash1622000

[14] C Bertolotto P Abbe T J Hemesath et al ldquoMicrophthalmiagene product as a signal transducer in cAMP-induced differen-tiation of melanocytesrdquo Journal of Cell Biology vol 142 no 3pp 827ndash835 1998

[15] C Gaggioli R Busca P Abbe J-P Ortonne and R Bal-lotti ldquoMicrophthalmia-associated transcription factor (MITF)is required but is not sufficient to induce the expression ofmelanogenic genesrdquo Pigment Cell Research vol 16 no 4 pp374ndash382 2003

[16] K S Hoek N C Schlegel O M Eichhoff et al ldquoNovel MITFtargets identified using a two-step DNA microarray strategyrdquoPigment Cell amp Melanoma Research vol 21 no 6 pp 665ndash6762008

[17] F Vetrini A Auricchio J Du et al ldquoThe microphthalmiatranscription factor (Mitf) controls expression of the ocularalbinism type 1 gene link between melanin synthesis andmelanosome biogenesisrdquo Molecular and Cellular Biology vol24 no 15 pp 6550ndash6559 2004

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 15

[18] C Bertolotto K Bille J-P Ortonne andR Ballotti ldquoRegulationof tyrosinase gene expression by cAMP in B16 melanoma cellsinvolves two CATGTG motifs surrounding the TATA boximplication of themicrophthalmia gene productrdquo Journal of CellBiology vol 134 no 3 pp 747ndash755 1996

[19] C Bertolotto R Busca P Abbe et al ldquoDifferent cis-actingelements are involved in the regulation of TRP1 and TRP2promoter activities by cyclic AMP pivotal role of M boxes(GTCATGTGCT) and of microphthalmiardquoMolecular and Cel-lular Biology vol 18 no 2 pp 694ndash702 1998

[20] C Chiaverini L Beuret E Flori et al ldquoMicrophthalmia-associated transcription factor regulates RAB27A gene expres-sion and controls melanosome transportrdquo Journal of BiologicalChemistry vol 283 no 18 pp 12635ndash12642 2008

[21] T Passeron P Bahadoran C Bertolotto et al ldquoCyclic AMPpro-motes a peripheral distribution of melanosomes and stimulatesmelanophilinSlac2-a actin associationrdquo FASEB Journal vol 18no 9 pp 989ndash991 2004

[22] F P Noonan T Otsuka S Bang M R Anver and G Mer-lino ldquoAccelerated ultraviolet radiation-induced carcinogenesisin hepatocyte growth factorscatter factor transgenic micerdquoCancer Research vol 60 no 14 pp 3738ndash3743 2000

[23] A Van Schanke M J Jongsma R Bisschop G M C A LVan Venrooij H Rebel and F R De Gruijl ldquoSingle UVBoverexposure stimulates melanocyte proliferation in murineskin in contrast to fractionated or UVA-1 exposurerdquo Journal ofInvestigative Dermatology vol 124 no 1 pp 241ndash247 2005

[24] G J Walker M G Kimlin E Hacker et al ldquoMurine neonatalmelanocytes exhibit a heightened proliferative response toultraviolet radiation and migrate to the epidermal basal layerrdquoJournal of Investigative Dermatology vol 129 no 1 pp 184ndash1932009

[25] T Hirobe ldquoRole of keratinocyte-derived factors involved inregulating the proliferation and differentiation of mammalianepidermalmelanocytesrdquo Pigment Cell Research vol 18 no 1 pp2ndash12 2005

[26] R Busca P Abbe F Mantoux et al ldquoRas mediates the cAMP-dependent activation of extracellular signal-regulated kinases(ERKs) in melanocytesrdquo EMBO Journal vol 19 no 12 pp2900ndash2910 2000

[27] G G McGill M Horstmann H R Widlund et al ldquoBcl2regulation by the melanocyte master regulator Mitf modulateslineage survival and melanoma cell viabilityrdquo Cell vol 109 no6 pp 707ndash718 2002

[28] R Haqa S Yokoyamab E B Hawryluk et al ldquoBCL2A1 is alineage-specific antiapoptotic melanoma oncogene that confersresistance to BRAF inhibitionrdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no11 pp 4321ndash4326 2013

[29] J N Dynek S M Chan J Liu J Zha W J Fairbrother andD Vucic ldquoMicrophthalmia-associated transcription factor isa critical transcriptional regulator of melanoma inhibitor ofapoptosis in melanomasrdquo Cancer Research vol 68 no 9 pp3124ndash3132 2008

[30] T Strub S Giuliano T Ye et al ldquoEssential role of microph-thalmia transcription factor for DNA replication mitosis andgenomic stability in melanomardquo Oncogene vol 30 no 20 pp2319ndash2332 2011

[31] M F Holick ldquoSunlight and vitamin D for bone health and pre-vention of autoimmune diseases cancers and cardiovasculardiseaserdquoThe American Journal of Clinical Nutrition vol 80 no6 Supplement pp 1678Sndash1688S 2004

[32] M F Holick ldquoVitamin D importance in the prevention of can-cers type 1 diabetes heart disease and osteoporosisrdquo AmericanJournal of Clinical Nutrition vol 79 no 3 pp 362ndash371 2004

[33] J A Curtin J Fridlyand T Kageshita et al ldquoDistinct setsof genetic alterations in melanomardquo New England Journal ofMedicine vol 353 no 20 pp 2135ndash2147 2005

[34] S Krengel A Hauschild and T Schafer ldquoMelanoma riskin congenital melanocytic naevi a systematic reviewrdquo BritishJournal of Dermatology vol 155 no 1 pp 1ndash8 2006

[35] C J Hussussian J P Struewing A M Goldstein et alldquoGermline p16 mutations in familial melanomardquo Nature Genet-ics vol 8 no 1 pp 15ndash21 1994

[36] L Zuo J Weger Q Yang et al ldquoGermline mutations in thep16(INK4a) binding domain of CDK4 in familial melanomardquoNature Genetics vol 12 no 1 pp 97ndash99 1996

[37] B Bressac-de-Paillerets M-F Avril A Chompret and FDemenais ldquoGenetic and environmental factors in cutaneousmalignant melanomardquo Biochimie vol 84 no 1 pp 67ndash74 2002

[38] M C Fargnoli S Gandini K Peris P Maisonneuve and SRaimondi ldquoMC1R variants increase melanoma risk in familieswith CDKN2Amutations ameta-analysisrdquo European Journal ofCancer vol 46 no 8 pp 1413ndash1420 2010

[39] C Bertolotto F Lesueur S Giuliano et al ldquoA SUMOylation-defective MITF germline mutation predisposes to melanomaand renal carcinomardquoNature vol 480 no 7375 pp 94ndash98 2011

[40] P Ghiorzo L Pastorino P Queirolo et al ldquoPrevalence of theE318K MITF germline mutation in Italian melanoma patientsassociations with histological subtypes and family cancer his-toryrdquoPigment Cell ampMelanomaResearch vol 26 no 2 pp 259ndash262 2013

[41] R A Sturm C Fox P McClenahan et al ldquoPhenotypiccharacterization of nevus and tumor patterns in MITF E318Kmutation carrier melanoma patientsrdquo Journal of InvestigativeDermatology 2013

[42] S Yokoyama S L Woods G M Boyle et al ldquoA novel recur-rent mutation in MITF predisposes to familial and sporadicmelanomardquo Nature vol 480 no 7375 pp 99ndash103 2011

[43] P T Bradford D M Freedman A M Goldstein and MA Tucker ldquoIncreased risk of second primary cancers after adiagnosis of melanomardquo Archives of Dermatology vol 146 no3 pp 265ndash272 2010

[44] F P Noonan J A Recio H Takayama et al ldquoNeonatal sunburnand melanoma in micerdquo Nature vol 413 no 6853 pp 271ndash2722001

[45] J L Bulliard ldquoSite-specific risk of cutaneous malignantmelanoma and pattern of sun exposure in New ZealandrdquoInternational Journal of Cancer vol 85 no 5 pp 627ndash632 2000

[46] S Mouret C Philippe J Gracia-Chantegrel et al ldquoUVA-induced cyclobutane pyrimidine dimers in DNA a direct pho-tochemical mechanismrdquo Organic and Biomolecular Chemistryvol 8 no 7 pp 1706ndash1711 2010

[47] P Filipe P Morliere J N Silva et al ldquoPlasma lipoproteinsas mediators of the oxidative stress induced by UV light inhuman skin a review of biochemical and biophysical studiesonmechanisms of apolipoprotein alteration lipid peroxidationand associated skin cell responsesrdquo Oxidative Medicine andCellular Longevity vol 2013 Article ID 285825 11 pages 2013

[48] E C De Fabo F P Noonan T Fears and GMerlino ldquoUltravio-let B but not ultraviolet A radiation initiates melanomardquoCancerResearch vol 64 no 18 pp 6372ndash6376 2004

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

16 Scientifica

[49] A R Lehmann D McGibbon and M Stefanini ldquoXerodermapigmentosumrdquo Orphanet Journal of Rare Diseases vol 6 no 1article 70 2011

[50] A Kauffmann F Rosselli V Lazar et al ldquoHigh expressionof DNA repair pathways is associated with metastasis inmelanoma patientsrdquoOncogene vol 27 no 5 pp 565ndash573 2008

[51] G Giglia-Mari and A Sarasin ldquoTP53 mutations in human skincancersrdquo Human Mutation vol 21 no 3 pp 217ndash228 2003

[52] C Greenman P Stephens R Smith et al ldquoPatterns of somaticmutation in human cancer genomesrdquoNature vol 446 no 7132pp 153ndash158 2007

[53] E D Pleasance R K Cheetham P J Stephens et al ldquoAcomprehensive catalogue of somatic mutations from a humancancer genomerdquo Nature vol 463 no 7278 pp 191ndash196 2010

[54] K C Cheng D S Cahill H Kasai S Nishimura and L ALoeb ldquo8-Hydroxyguanine an abundant form of oxidative DNAdamage causes G rarr T and A rarr C substitutionsrdquo Journal ofBiological Chemistry vol 267 no 1 pp 166ndash172 1992

[55] M F Berger E Hodis T P Heffernan et al ldquoMelanoma genomesequencing reveals frequent PREX2 mutationsrdquo Nature vol485 no 7399 pp 502ndash506 2012

[56] E Hodis I R Watson G V Kryukov et al ldquoA landscape ofdrivermutations inmelanomardquoCell vol 150 no 2 pp 251ndash2632012

[57] M Krauthammer Y Kong B H Ha et al ldquoExome sequencingidentifies recurrent somatic RAC1 mutations in melanomardquoNature Genetics vol 44 no 9 pp 1006ndash1014 2012

[58] D F Easton and R A Eeles ldquoGenome-wide association studiesin cancerrdquo Human Molecular Genetics vol 17 no 2 pp R109ndashR115 2008

[59] P D P Pharoah A Antoniou M Bobrow R L Zimmern DF Easton and B A J Ponder ldquoPolygenic susceptibility to breastcancer and implications for preventionrdquoNature Genetics vol 31no 1 pp 33ndash36 2002

[60] K Laud C Marian M F Avril et al ldquoComprehensive analysisof CDKN2A (p16INK4Ap14ARF) and CDKN2B genes in 53melanoma index cases considered to be at heightened risk ofmelanomardquo Journal of Medical Genetics vol 43 no 1 pp 39ndash47 2006

[61] J F Thompson R A Scolyer and R F Kefford ldquoCutaneousmelanoma in the era of molecular profilingrdquo The Lancet vol374 no 9687 pp 362ndash365 2009

[62] A A Nelson and H Tsao ldquoMelanoma and geneticsrdquo Clinics inDermatology vol 27 no 1 pp 46ndash52 2009

[63] N P Pavletich ldquoMechanisms of cyclin-dependent kinase regu-lation structures of Cdks their cyclin activators and Cip andINK4 inhibitorsrdquo Journal of Molecular Biology vol 287 no 5pp 821ndash828 1999

[64] D C Bennett ldquoHow to make a melanoma what do we know ofthe primary clonal eventsrdquo Pigment Cell ampMelanomaResearchvol 21 no 1 pp 27ndash38 2008

[65] V C Gray-Schopfer S C Cheong H Chong et al ldquoCellularsenescence in naevi and immortalisation in melanoma a rolefor p16rdquo British Journal of Cancer vol 95 no 4 pp 496ndash5052006

[66] E V Sviderskaya V C Gray-Schopfer S P Hill et alldquop16cyclin-dependent kinase inhibitor 2A deficiency in humanmelanocyte senescence apoptosis and immortalization pos-sible implications for melanoma progressionrdquo Journal of theNational Cancer Institute vol 95 no 10 pp 723ndash732 2003

[67] M Castellano B G Gabrielli C J Hussussian N C Dracopoliand N K Hayward ldquoRestoration of CDKN2A into melanomacells induces morphologic changes and reduction in growthrate but not anchorage-independent growth reversalrdquo Journalof Investigative Dermatology vol 109 no 1 pp 61ndash68 1997

[68] J Ackermann M Frutschi K Kaloulis T McKee A Trumppand F Beermann ldquoMetastasizing melanoma formation causedby expression of activated N-RasQ61K on an INK4a-deficientbackgroundrdquo Cancer Research vol 65 no 10 pp 4005ndash40112005

[69] H Rizos S Puig C Badenas et al ldquoA melanoma-associatedgermline mutation in exon 1120573 inactivates p14ARFrdquo Oncogenevol 20 no 39 pp 5543ndash5547 2001

[70] C Hewitt C LWu G Evans et al ldquoGermlinemutation of ARFin a melanoma kindredrdquoHumanMolecular Genetics vol 11 no11 pp 1273ndash1279 2002

[71] J A Randerson-Moor M Harland S Williams et al ldquoAgermline deletion of p14ARF but not CDKN2A in a melanoma-neural system tumour syndrome familyrdquo Human MolecularGenetics vol 10 no 1 pp 55ndash62 2001

[72] D E Freedberg S H Rigas J Russak et al ldquoFrequent p16-independent inactivation of p14ARF in human melanomardquoJournal of the National Cancer Institute vol 100 no 11 pp 784ndash795 2008

[73] L Ha T Lchikawa M Anver et al ldquoARF functions as amelanoma tumor suppressor by inducing p53-independentsenescencerdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 26 pp 10968ndash109732007

[74] N E Sharpless K Kannan J Xu M W Bosenberg and LChin ldquoBoth products of the mouse INK4aARF locus suppressmelanoma formation in vivordquo Oncogene vol 22 no 32 pp5055ndash5059 2003

[75] M Daniotti M Oggionni T Ranzani et al ldquoBRAF alterationsare associated with complex mutational profiles in malignantmelanomardquo Oncogene vol 23 no 35 pp 5968ndash5977 2004

[76] N SoufirM-F Avril A Chompret et al ldquoPrevalence of p16 andCDK4 germline mutations in 48 melanoma-prone families inFrance The French Familial Melanoma Study Grouprdquo HumanMolecular Genetics vol 7 no 2 pp 209ndash216 1998

[77] S G Rane S C Cosenza R V Mettus and E P ReddyldquoGerm line transmission of the Cdk4R24C mutation facilitatestumorigenesis and escape from cellular senescencerdquo Molecularand Cellular Biology vol 22 no 2 pp 644ndash656 2002

[78] R Chawla J A Procknow R V Tantravahi J S Khurana JLitvin and E P Reddy ldquoCooperativity of Cdk4R24C and Rasin melanoma developmentrdquo Cell Cycle vol 9 no 16 pp 3305ndash3314 2010

[79] R Sotillo J F Garcıa S Ortega et al ldquoInvasive melanoma inCdk4-targeted micerdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 98 no 23 pp 13312ndash13317 2001

[80] E Steingrımsson N G Copeland and N A JenkinsldquoMelanocytes and the Microphthalmia transcription factornetworkrdquo Annual Review of Genetics vol 38 pp 365ndash411 2004

[81] Y CheliMOhanna R Ballotti andC Bertolotto ldquoFifteen-yearquest for microphthalmia-associated transcription factor targetgenesrdquo Pigment Cell amp Melanoma Research vol 23 no 1 pp27ndash40 2010

[82] L A Garraway H R Widlund M A Rubin et al ldquoIntegrativegenomic analyses identify MITF as a lineage survival oncogene

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 17

amplified in malignant melanomardquo Nature vol 436 no 7047pp 117ndash122 2005

[83] S Ugurel R Houben D Schrama et al ldquoMicrophthalmia-associated transcription factor gene amplification in metastaticmelanoma is a prognostic marker for patient survival but nota predictive marker for chemosensitivity and chemotherapyresponserdquo Clinical Cancer Research vol 13 no 21 pp 6344ndash6350 2007

[84] S Carreira J Goodall L Denat et al ldquoMitf regulation of Dia1controls melanoma proliferation and invasivenessrdquo Genes andDevelopment vol 20 no 24 pp 3426ndash3439 2006

[85] Y Cheli S Guiliano T Botton et al ldquoMitf is the key molecularswitch between mouse or human melanoma initiating cells andtheir differentiated progenyrdquoOncogene vol 30 no 20 pp 2307ndash2318 2011

[86] Y Cheli S Giuliano N Fenouille et al ldquoHypoxia and MITFcontrol metastatic behaviour in mouse and human melanomacellsrdquo Oncogene vol 31 no 19 pp 2461ndash2470 2012

[87] K S Hoek and C R Goding ldquoCancer stem cells versusphenotype-switching in melanomardquo Pigment Cell amp MelanomaResearch vol 23 no 6 pp 746ndash759 2010

[88] S Carreira J Goodall I Aksan et al ldquoMitf cooperates withRb1 and activates p21Cip1 expression to regulate cell cycleprogressionrdquo Nature vol 433 no 7027 pp 764ndash769 2005

[89] S Giuliano Y Cheli M Ohanna et al ldquoMicrophthalmia-associated transcription factor controls the DNA damageresponse and a lineage-specific senescence program in mela-nomasrdquo Cancer Research vol 70 no 9 pp 3813ndash3822 2010

[90] S Giuliano M Ohanna R Ballotti and C BertolottoldquoAdvances inmelanoma senescence and potential clinical appli-cationrdquo Pigment Cell amp Melanoma Research vol 24 no 2 pp295ndash308 2011

[91] MOhanna S Giuliano C Bonet et al ldquoSenescent cells developa PARP-1 and nuclear factor-120581B-associated secretome (PNAS)rdquoGenes and Development vol 25 no 12 pp 1245ndash1261 2011

[92] T J Hemesath E R Price C Takemoto T Badalian and DE Fisher ldquoMAP kinase links the transcription factor Microph-thalmia to c-Kit signalling in melanocytesrdquoNature vol 391 no6664 pp 298ndash301 1998

[93] W Xu L Gong M M Haddad et al ldquoRegulation ofmicrophthalmia-associated transcription factor MITF proteinlevels by association with the ubiquitin-conjugating enzymehUBC9rdquo Experimental Cell Research vol 255 no 2 pp 135ndash1432000

[94] K AWilkinson and JM Henley ldquoMechanisms regulation andconsequences of protein SUMOylationrdquo Biochemical Journalvol 428 no 2 pp 133ndash145 2010

[95] J Du H R Widlund M A Horstmann et al ldquoCritical role ofCDK2 for melanoma growth linked to its melanocyte-specifictranscriptional regulation by MITFrdquo Cancer Cell vol 6 no 6pp 565ndash576 2004

[96] L Beuret E Flori C Denoyelle et al ldquoUp-regulation of METexpression by 120572-melanocyte-stimulating hormone and MITFallows hepatocyte growth factor to protect melanocytes andmelanoma cells fromapoptosisrdquo Journal of Biological Chemistryvol 282 no 19 pp 14140ndash14147 2007

[97] R Busca E Berra C Gaggioli et al ldquoHypoxia-inducible factor1120572 is a new target of microphthalmia-associated transcriptionfactor (MITF) in melanoma cellsrdquo Journal of Cell Biology vol170 no 1 pp 49ndash59 2005

[98] F Liu Y Fu and F L Meyskens Jr ldquoMiTF regulates cellularresponse to reactive oxygen species through transcriptionalregulation of APE-1Ref-1rdquo Journal of Investigative Dermatologyvol 129 no 2 pp 422ndash431 2009

[99] T J Hornyak S Jiang E A Guzman et al ldquoMitf dosage asa primary determinant of melanocyte survival after ultravioletirradiationrdquo Pigment Cell amp Melanoma Research vol 22 no 3pp 307ndash318 2009

[100] A J Miller J Du S Rowan C L Hershey H R Widlund andD E Fisher ldquoTranscriptional regulation of the melanoma prog-nostic marker melastatin (TRPM1) by MITF in melanocytesand melanomardquo Cancer Research vol 64 no 2 pp 509ndash5162004

[101] R Haq J Shoag P Andreu-Perez et al ldquoOncogenic BRAFregulates oxidative metabolism via PGC1alpha and MITFrdquoCancer Cell vol 23 no 3 pp 302ndash315 2013

[102] F Vazquez J-H Lim H Chim et al ldquoPGC1alpha expressiondefines a subset of human melanoma tumors with increasedmitochondrial capacity and resistance to oxidative stressrdquoCancer Cell vol 23 no 3 pp 287ndash301 2013

[103] C Handschin and B M Spiegelman ldquoPeroxisome proliferator-activated receptor 120574 coactivator 1 coactivators energy home-ostasis and metabolismrdquo Endocrine Reviews vol 27 no 7 pp728ndash735 2006

[104] J L Rees ldquoThemelanocortin 1 receptor (MC1R) more than justred hairrdquo Pigment Cell Research vol 13 no 3 pp 135ndash140 2000

[105] R A Sturm ldquoSkin colour and skin cancermdashMC1R the geneticlinkrdquoMelanoma Research vol 12 no 5 pp 405ndash416 2002

[106] C Kennedy J Ter Huurne M Berkhout et al ldquoMelanocortin 1receptor (MC1R) gene variants are associated with an increasedrisk for cutaneous melanoma which is largely independent ofskin type and hair colorrdquo Journal of Investigative Dermatologyvol 117 no 2 pp 294ndash300 2001

[107] J S Palmer D L Duffy N F Box et al ldquoMelanocortin-1receptor polymorphisms and risk of melanoma is the associ-ation explained solely by pigmentation phenotyperdquo AmericanJournal of Human Genetics vol 66 no 1 pp 176ndash186 2000

[108] P Valverde E Healy I Jackson J L Rees and A J ThodyldquoVariants of the melanocyte-stimulating hormone receptorgene are associated with red hair and fair skin in humansrdquoNature Genetics vol 11 no 3 pp 328ndash330 1995

[109] N F Box D L DuffyW Chen et al ldquoMC1R genotype modifiesrisk of melanoma in families segregating CDKN2AmutationsrdquoAmerican Journal of HumanGenetics vol 69 no 4 pp 765ndash7732001

[110] P A Van der Velden L A Sandkuijl W Bergman et alldquoMelanocortin-1 receptor variant R151C modifies melanomarisk in Dutch families with melanomardquo American Journal ofHuman Genetics vol 69 no 4 pp 774ndash779 2001

[111] M C Fargnoli K Pike R M Pfeiffer et al ldquoMC1R variantsincrease risk of melanomas harboring BRAF mutationsrdquo Jour-nal of Investigative Dermatology vol 128 no 10 pp 2485ndash24902008

[112] M T Landi J Bauer R M Pfeiffer et al ldquoMC1R germlinevariants confer risk for BRAF-mutant melanomardquo Science vol313 no 5786 pp 521ndash522 2006

[113] D F Gudbjartsson P Sulem S N Stacey et al ldquoASIP and TYRpigmentation variants associate with cutaneous melanoma andbasal cell carcinomardquo Nature Genetics vol 40 no 7 pp 886ndash891 2008

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

18 Scientifica

[114] L P Fernandez R L Milne G Pita et al ldquoSLC45A2 a novelmalignant melanoma-associated generdquo Human Mutation vol29 no 9 pp 1161ndash1167 2008

[115] MKvaskoffD CWhiteman Z Z Zhao et al ldquoPolymorphismsin nevus-associated genes MTAP PLA2G6 and IRF4 andthe risk of invasive cutaneous melanomardquo Twin Research andHuman Genetics vol 14 no 5 pp 422ndash432 2011

[116] S Horn A Figl P S Rachakonda et al ldquoTERT promotermutations in familial and sporadicmelanomardquo Science vol 339no 6122 pp 959ndash961 2013

[117] X Wei V Walia J C Lin et al ldquoExome sequencing identifiesGRIN2A as frequently mutated in melanomardquoNature Geneticsvol 43 no 5 pp 442ndash448 2011

[118] T D Prickett N S Agrawal X Wei et al ldquoAnalysis of thetyrosine kinome in melanoma reveals recurrent mutations inERBB4rdquo Nature Genetics vol 41 no 10 pp 1127ndash1132 2009

[119] L H Palavalli T D Prickett J RWunderlich et al ldquoAnalysis ofthe matrix metalloproteinase family reveals that MMP8 is oftenmutated in melanomardquo Nature Genetics vol 41 no 5 pp 518ndash520 2009

[120] R Ghai M Mobli S J Norwood et al ldquoPhox homology band41ezrinradixinmoesin-like proteins function as molecularscaffolds that interact with cargo receptors and Ras GTPasesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 19 pp 7763ndash7768 2011

[121] M Cully J Shiu R P Piekorz W J Muller S J Done and TW Mak ldquoTransforming acidic coiled coil 1 promotes transfor-mation andmammary tumorigenesisrdquoCancer Research vol 65no 22 pp 10363ndash10370 2005

[122] P M Pollock U L Harper K S Hansen et al ldquoHigh frequencyof BRAF mutations in nevirdquo Nature Genetics vol 33 no 1 pp19ndash20 2003

[123] I Yeh A von Deimling B C Bastian et al ldquoClonal BRAFmutations in melanocytic nevi and initiating role of BRAF inmelanocytic neoplasiardquo Journal of the National Cancer Institutevol 105 no 12 pp 917ndash919 2013

[124] Y Chudnovsky A E Adams P B Robbins Q Lin and P AKhavari ldquoUse of human tissue to assess the oncogenic activityofmelanoma-associatedmutationsrdquoNatureGenetics vol 37 no7 pp 745ndash749 2005

[125] B Bedogni and M B Powell ldquoHypoxia melanocytes andmelanomamdashsurvival and tumor development in the permissivemicroenvironment of the skinrdquo Pigment Cell amp MelanomaResearch vol 22 no 2 pp 166ndash174 2009

[126] S S Dadras ldquoMolecular diagnostics in Melanoma currentstatus and perspectivesrdquo Archives of Pathology and LaboratoryMedicine vol 135 no 7 pp 860ndash869 2011

[127] C Denoyelle G Abou-Rjaily V Bezrookove et al ldquoAnti-oncogenic role of the endoplasmic reticulum differentiallyactivated by mutations in the MAPK pathwayrdquo Nature CellBiology vol 8 no 10 pp 1053ndash1063 2006

[128] C Michaloglou L C W Vredeveld M S Soengas et alldquoBRAFE600-associated senescence-like cell cycle arrest ofhuman naevirdquo Nature vol 436 no 7051 pp 720ndash724 2005

[129] D Dankort D P Curley R A Cartlidge et al ldquoBrafV600Ecooperates with Pten loss to induce metastatic melanomardquoNature Genetics vol 41 no 5 pp 544ndash552 2009

[130] N Dhomen J S Reis-Filho S da Rocha Dias et al ldquoOncogenicbraf induces melanocyte senescence and melanoma in micerdquoCancer Cell vol 15 no 4 pp 294ndash303 2009

[131] W E Damsky D P Curley M Santhanakrishnan et al ldquo120573-catenin signaling controls metastasis in braf-activated pten-deficient melanomasrdquo Cancer Cell vol 20 no 6 pp 741ndash7542011

[132] D C Whiteman W J Pavan and B C Bastian ldquoThemelanomas a synthesis of epidemiological clinical histopatho-logical genetic and biological aspects supporting distinctsubtypes causal pathways and cells of originrdquo Pigment Cell ampMelanoma Research vol 24 no 5 pp 879ndash897 2011

[133] U Banerji A Affolter I Judson R Marais and P WorkmanldquoBRAF and NRAS mutations in melanoma potential rela-tionships to clinical response to HSP90 inhibitorsrdquo MolecularCancer Therapeutics vol 7 no 4 pp 737ndash739 2008

[134] J A Ellerhorst V R Greene S Ekmekcioglu C LWarnekeMM Johnson and C P Cooke ldquoClinical correlates of NRAS andBRAFmutations in primary humanmelanomardquoClinical CancerResearch vol 17 no 2 pp 229ndash235 2011

[135] M C R E Van Dijk M R Bernsen and D J Ruiter ldquoAnalysisof mutations in B-RAF N-RAS and H-RAS genes in thedifferential diagnosis of Spitz nevus and spitzoid melanomardquoAmerican Journal of Surgical Pathology vol 29 no 9 pp 1145ndash1151 2005

[136] H Davies G R Bignell C Cox et al ldquoMutations of the BRAFgene in human cancerrdquo Nature vol 417 no 6892 pp 949ndash9542002

[137] J A Jakob R L Bassett Jr C S Ng et al ldquoNRAS muta-tion status is an independent prognostic factor in metastaticmelanomardquo Cancer vol 118 no 16 pp 4014ndash4023 2012

[138] B Jovanovic S Egyhazi M Eskandarpour et al ldquoCoexistingNRAS and BRAF mutations in primary familial melanomaswith specific CDKN2A germline alterationsrdquo Journal of Inves-tigative Dermatology vol 130 no 2 pp 618ndash620 2010

[139] C Wellbrock S Rana H Paterson H Pickersgill T Brum-melkamp and R Marais ldquoOncogenic BRAF regulates mela-noma proliferation through the lineage specific factor MITFrdquoPLoS ONE vol 3 no 7 Article ID e2734 2008

[140] B Govindarajan X Bai C Cohen et al ldquoMalignant transfor-mation of melanocytes to melanoma by constitutive activationof mitogen-activated protein kinase kinase (MAPKK) signal-ingrdquo Journal of Biological Chemistry vol 278 no 11 pp 9790ndash9795 2003

[141] B Jansen H Schlagbauer-Wadl H Kahr et al ldquoNovel Rasantagonist blocks humanmelanoma growthrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 24 pp 14019ndash14024 1999

[142] S R Hingorani M A Jacobetz G P Robertson M Herlynand D A Tuveson ldquoSuppression of BRAFV599E in humanmelanoma abrogates transformationrdquo Cancer Research vol 63no 17 pp 5198ndash5202 2003

[143] K P Hoeflich D C Gray M T Eby et al ldquoOncogenic BRAFis required for tumor growth and maintenance in melanomamodelsrdquo Cancer Research vol 66 no 2 pp 999ndash1006 2006

[144] A Sharma M A Tran S Liang et al ldquoTargeting mitogen-activated protein kinaseextracellular signal-regulated kinasekinase in the mutant (V600E) B-Raf signaling cascade effec-tively inhibitsmelanoma lungmetastasesrdquoCancer Research vol66 no 16 pp 8200ndash8209 2006

[145] J Caramel E Papadogeorgakis L Hill et al ldquoA switch in theexpression of embryonic EMT-inducers drives the Develop-ment of Malignant Melanomardquo Cancer Cell vol 24 no 4 pp466ndash480 2013

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 19

[146] M Chraybi I Abd Alsamad C Copie-Bergman et al ldquoOnco-gene abnormalities in a series of primary melanomas of thesinonasal tract NRAS mutations and cyclin D1 amplificationare more frequent than KIT or BRAF mutationsrdquo HumanPathology vol 44 no 9 pp 1902ndash1911 2013

[147] E R Sauter U-C Yeo A Von Stemm et al ldquoCyclin D1 is acandidate oncogene in cutaneous melanomardquo Cancer Researchvol 62 no 11 pp 3200ndash3206 2002

[148] K S M Smalley M Lioni M D Palma et al ldquoIncreased cyclinD1 expression can mediate BRAF inhibitor resistance in BRAFV600E-mutated melanomasrdquo Molecular Cancer Therapeuticsvol 7 no 9 pp 2876ndash2883 2008

[149] K Trunzer et al ldquoPharmacodynamic effects and mecha-nisms of resistance to vemurafenib in patients with metastaticmelanomardquo Journal of Clinical Oncology vol 31 no 14 pp 1767ndash1774 2013

[150] K V Bhatt L S Spofford G AramMMcMullen K Pumigliaand A E Aplin ldquoAdhesion control of cyclin D1 and p27Kip1levels is deregulated in melanoma cells through BRAF-MEK-ERK signalingrdquo Oncogene vol 24 no 21 pp 3459ndash3471 2005

[151] V Alexeev and K Yoon ldquoDistinctive role of the cKit receptortyrosine kinase signaling in mammalian melanocytesrdquo Journalof Investigative Dermatology vol 126 no 5 pp 1102ndash1110 2006

[152] S Nishikawa M Kusakabe K Yoshinaga et al ldquoIn uteromanipulation of coat color formation by a monoclonal anti-c-kit antibody two distinct waves of c-kit-dependency duringmelanocyte developmentrdquo EMBO Journal vol 10 no 8 pp2111ndash2118 1991

[153] M Okura H Maeda S Nishikawa andM Mizoguchi ldquoEffectsof monoclonal anti-c-Kit antibody (ACK2) on melanocytes innewborn micerdquo Journal of Investigative Dermatology vol 105no 3 pp 322ndash328 1995

[154] F Janku J Novotny I Julis et al ldquoKIT receptor is expressedin more than 50 of early-stage malignant melanoma aretrospective study of 261 patientsrdquoMelanoma Research vol 15no 4 pp 251ndash256 2005

[155] A K Mobley et al ldquoDriving transcriptional regulators inmelanoma metastasisrdquo Cancer and Metastasis Reviews vol 31no 3-4 pp 621ndash632 2012

[156] M Bar-Eli ldquoRole of AP-2 in tumor growth and metastasis ofhuman melanomardquo Cancer and Metastasis Reviews vol 18 no3 pp 377ndash385 1999

[157] L A McPherson A V Loktev and R J Weigel ldquoTumor sup-pressor activity of AP2120572 mediated through a direct interactionwith p53rdquo Journal of Biological Chemistry vol 277 no 47 pp45028ndash45033 2002

[158] C R Antonescu K J Busam T D Francone et al ldquoL576PKIT mutation in anal melanomas correlates with KIT proteinexpression and is sensitive to specific kinase inhibitionrdquo Inter-national Journal of Cancer vol 121 no 2 pp 257ndash264 2007

[159] C Beadling E Jacobson-Dunlop F S Hodi et al ldquoKIT genemutations and copy number in melanoma subtypesrdquo ClinicalCancer Research vol 14 no 21 pp 6821ndash6828 2008

[160] J A Curtin K Busam D Pinkel and B C Bastian ldquoSomaticactivation of KIT in distinct subtypes of melanomardquo Journal ofClinical Oncology vol 24 no 26 pp 4340ndash4346 2006

[161] C Willmore-Payne J A Holden S Hirschowitz and L JLayfield ldquoBRAF and c-kit gene copy number in mutation-positive malignant melanomardquo Human Pathology vol 37 no5 pp 520ndash527 2006

[162] S Huang D JeanM LucaM A Tainsky andM Bar-Eli ldquoLossof AP-2 results in downregulation of c-KIT and enhancement ofmelanoma tumorigenicity and metastasisrdquo EMBO Journal vol17 no 15 pp 4358ndash4369 1998

[163] K S M Smalley R Contractor T K Nguyen et al ldquoIdenti-fication of a novel subgroup of melanomas with KITcyclin-dependent kinase-4 overexpressionrdquo Cancer Research vol 68no 14 pp 5743ndash5752 2008

[164] K H Vousden and C Prives ldquoBlinded by the light the growingcomplexity of p53rdquo Cell vol 137 no 3 pp 413ndash431 2009

[165] L A Donehower M Harvey B L Slagle et al ldquoMice deficientfor p53 are developmentally normal but susceptible to sponta-neous tumoursrdquo Nature vol 356 no 6366 pp 215ndash221 1992

[166] N Ibrahim and F G Haluska ldquoMolecular pathogenesis of cuta-neous melanocytic neoplasmsrdquo Annual Review of Pathologyvol 4 pp 551ndash579 2009

[167] N Bardeesy B C Bastian A Hezel D Pinkel R A DePinhoand L Chin ldquoDual inactivation of RB and p53 pathways in RAS-inducedmelanomasrdquoMolecular andCellular Biology vol 21 no6 pp 2144ndash2153 2001

[168] M Dovey R M White and L I Zon ldquoOncogenic NRAScooperates with p53 loss to generate melanoma in zebrafishrdquoZebrafish vol 6 no 4 pp 397ndash404 2009

[169] V K Goel N Ibrahim G Jiang et al ldquoMelanocytic nevus-likehyperplasia and melanoma in transgenic BRAFV600E micerdquoOncogene vol 28 no 23 pp 2289ndash2298 2009

[170] E E Patton H R Widlund J L Kutok et al ldquoBRAFmutationsare sufficient to promote nevi formation and cooperate with p53in the genesis of melanomardquo Current Biology vol 15 no 3 pp249ndash254 2005

[171] M S Soengas P Capodieci D Polsky et al ldquoInactivation of theapoptosis effector Apaf-1 in malignant melanomardquo Nature vol409 no 6817 pp 207ndash211 2001

[172] H Yu R McDaid J Lee et al ldquoThe role of BRAF mutation andp53 inactivation during transformation of a subpopulation ofprimary human melanocytesrdquo American Journal of Pathologyvol 174 no 6 pp 2367ndash2377 2009

[173] V Muthusamy C Hobbs C Nogueira et al ldquoAmplification ofCDK4 and MDM2 in malignant melanomardquo Genes Chromo-somes and Cancer vol 45 no 5 pp 447ndash454 2006

[174] A Gembarska F Luciani C Fedele et al ldquoMDM4 is a keytherapeutic target in cutaneous melanomardquo Nature Medicinevol 18 no 8 pp 1239ndash1247 2012

[175] B Bedogni S M Welford D S Cassarino B J Nickoloff A JGiaccia and M B Powell ldquoThe hypoxic microenvironment ofthe skin contributes to Akt-mediated melanocyte transforma-tionrdquo Cancer Cell vol 8 no 6 pp 443ndash454 2005

[176] M Kaluzova S Kaluz M I Lerman and E J StanbridgeldquoDNA damage is a prerequisite for p53-mediated proteasomaldegradation of HIF-1120572 in hypoxic cells and downregulationof the hypoxia marker carbonic anhydrase IXrdquo Molecular andCellular Biology vol 24 no 13 pp 5757ndash5766 2004

[177] N H Kim H S Kim X-Y Li et al ldquoA p53miRNA-34 axisregulates Snail1-dependent cancer cell epithelial-mesenchymaltransitionrdquo Journal of Cell Biology vol 195 no 3 pp 417ndash4332011

[178] T Kim A Veronese F Pichiorri et al ldquop53 regulates epithelial-mesenchymal transition through microRNAs targeting ZEB1and ZEB2rdquo Journal of Experimental Medicine vol 208 no 5pp 875ndash883 2011

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

20 Scientifica

[179] H Tsao M B Atkins and A J Sober ldquoManagement ofcutaneous melanomardquo New England Journal of Medicine vol351 no 10 pp 998ndash1042 2004

[180] J M Stahl M Cheung A Sharma N R Trivedi S Shan-mugam and G P Robertson ldquoLoss of PTEN promotes tumordevelopment inmalignantmelanomardquoCancer Research vol 63no 11 pp 2881ndash2890 2003

[181] J M Stahl A Sharma M Cheung et al ldquoDeregulated Akt3activity promotes development of malignant melanomardquo Can-cer Research vol 64 no 19 pp 7002ndash7010 2004

[182] A Y Shull A Latham-Schwark P Ramasamy et al ldquoNovelsomatic mutations to PI3K pathway genes in metastaticmelanomardquo PLoS ONE vol 7 no 8 Article ID e43369 2012

[183] V K Goel A J F Lazar C L Warneke M S Redston and FG Haluska ldquoExamination of mutations in BRAF NRAS andPTEN in primary cutaneousmelanomardquo Journal of InvestigativeDermatology vol 126 no 1 pp 154ndash160 2006

[184] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[185] P Wang X Wang P Wu et al ldquoGM3 upregulation of matrixmetalloproteinase-9 possibly through PI3K AKT RICTORRHOGDI-2 and TNF-a pathways in mouse melanoma B16cellsrdquo Advances in Experimental Medicine and Biology vol 705pp 335ndash348 2011

[186] I Stamenkovic ldquoMatrix metalloproteinases in tumor invasionand metastasisrdquo Seminars in Cancer Biology vol 10 no 6 pp415ndash433 2000

[187] A Biddle and I C Mackenzie ldquoCancer stem cells and EMT incarcinomardquo Cancer and Metastasis Reviews vol 31 no 1-2 pp285ndash293 2012

[188] D Olmeda M Jorda H Peinado A Fabra and A Cano ldquoSnailsilencing effectively suppresses tumour growth and invasive-nessrdquo Oncogene vol 26 no 13 pp 1862ndash1874 2007

[189] H Peinado D Olmeda and A Cano ldquoSnail ZEB and bHLHfactors in tumour progression an alliance against the epithelialphenotyperdquo Nature Reviews Cancer vol 7 no 6 pp 415ndash4282007

[190] J P Thiery H Acloque R Y J Huang and M A NietoldquoEpithelial-mesenchymal transitions in development and dis-easerdquo Cell vol 139 no 5 pp 871ndash890 2009

[191] J Yang and R A Weinberg ldquoEpithelial-mesenchymal transi-tion at the crossroads of development and tumor metastasisrdquoDevelopmental Cell vol 14 no 6 pp 818ndash829 2008

[192] S R Alonso L Tracey P Ortiz et al ldquoA high-throughput studyin melanoma identifies epithelial-mesenchymal transition as amajor determinant of metastasisrdquo Cancer Research vol 67 no7 pp 3450ndash3460 2007

[193] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[194] K M Hajra D Y-S C David Y-S Chen and E R FearonldquoThe SLUG zinc-finger protein represses E-cadherin in breastcancerrdquo Cancer Research vol 62 no 6 pp 1613ndash1618 2002

[195] J Yang S AMani J LDonaher et al ldquoTwist amaster regulatorof morphogenesis plays an essential role in tumor metastasisrdquoCell vol 117 no 7 pp 927ndash939 2004

[196] P B Gupta C Kuperwasser J-P Brunet et al ldquoThemelanocytedifferentiation program predisposes tometastasis after neoplas-tic transformationrdquo Nature Genetics vol 37 no 10 pp 1047ndash1054 2005

[197] I K Bukholm J M Nesland and A L Borresen-Dale ldquoRe-expression of E-cadherin alpha-catenin and beta-catenin butnot of gamma-catenin in metastatic tissue from breast cancerpatients [seecomments]rdquo Journal of Pathology vol 190 no 1 pp15ndash19 2000

[198] M-Y Hsu F E Meier M Nesbit et al ldquoE-cadherin expressionin melanoma cells restores keratinocyte-mediated growth con-trol and down-regulates expression of invasion-related adhe-sion receptorsrdquo American Journal of Pathology vol 156 no 5pp 1515ndash1525 2000

[199] J E Kim E Leung B C Baguley andG J Finlay ldquoHeterogene-ity of expression of epithelial-mesenchymal transition markersin melanocytes and melanoma cell linesrdquo Frontiers in Geneticsvol 4 p 97 2013

[200] Y Ogawara S Kishishita T Obata et al ldquoAkt enhances Mdm2-mediated ubiquitination and degradation of p53rdquo Journal ofBiological Chemistry vol 277 no 24 pp 21843ndash21850 2002

[201] C-J Chang C-H Chao W Xia et al ldquoP53 regulates epithelial-mesenchymal transition and stem cell properties throughmod-ulating miRNAsrdquoNature Cell Biology vol 13 no 3 pp 317ndash3232011

[202] J I Yook X-Y Li I Ota et al ldquoA Wnt-Axin2-GSK3120573 cascaderegulates Snail1 activity in breast cancer cellsrdquo Nature CellBiology vol 8 no 12 pp 1398ndash1406 2006

[203] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[204] B P Zhou J Deng W Xia et al ldquoDual regulation of Snailby GSK-3120573-mediated phosphorylation in control of epithelial-mesenchymal transitionrdquo Nature Cell Biology vol 6 no 10 pp931ndash940 2004

[205] D Javelaud V Delmas M Moller et al ldquoStable overexpressionof Smad7 in human melanoma cells inhibits their tumorigenic-ity in vitro and in vivordquoOncogene vol 24 no 51 pp 7624ndash76292005

[206] J Massague ldquoTGFbeta in cancerrdquo Cell vol 134 no 2 pp 215ndash230 2008

[207] S Dennler J Andre I Alexaki et al ldquoInduction of sonichedgehog mediators by transforming growth factor-120573 smad3-dependent activation of Gli2 and Gli1 expression in vitro and invivordquo Cancer Research vol 67 no 14 pp 6981ndash6986 2007

[208] C Y Perrot C Gilbert V Marsaud A Postigo D Javelaudand AMauviel ldquoGLI2 cooperates with ZEB1 for transcriptionalrepression of CDH1 expression in human melanoma cellsrdquoPigment Cell amp Melanoma Research vol 26 no 6 pp 861ndash8732013

[209] C Y Perrot D Javelaud and A Mauviel ldquoInsights into thetransforming growth factor-beta signaling pathway in cuta-neousmelanomardquoAnnals of Dermatology vol 25 no 2 pp 135ndash144 2013

[210] C Y Logan and R Nusse ldquoTheWnt signaling pathway in devel-opment and diseaserdquo Annual Review of Cell and DevelopmentalBiology vol 20 pp 781ndash810 2004

[211] J Goodall S Martinozzi T J Dexter et al ldquoBrn-2 expressioncontrols melanoma proliferation and is directly regulated by 120573-cateninrdquoMolecular and Cellular Biology vol 24 no 7 pp 2915ndash2922 2004

[212] T-C He A B Sparks C Rago et al ldquoIdentification of c-MYCas a target of the APC pathwayrdquo Science vol 281 no 5382 pp1509ndash1512 1998

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Scientifica 21

[213] M Shtutman J Zhurinsky I Simcha et al ldquoThe cyclin D1 geneis a target of the 120573-cateninLEF-1 pathwayrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 10 pp 5522ndash5527 1999

[214] D L Rimm K Caca G Hu F B Harrison and E R FearonldquoFrequent nuclearcytoplasmic localization of 120573-catenin with-out exon 3 mutations in malignant melanomardquo AmericanJournal of Pathology vol 154 no 2 pp 325ndash329 1999

[215] J Reifenberger C B Knobbe M Wolter et al ldquoMoleculargenetic analysis of malignant melanomas for aberrations of thewnt signaling pathway genes CTNNB1 APC ICAT and BTRCrdquoInternational Journal of Cancer vol 100 no 5 pp 549ndash5562002

[216] V Delmas F Beermann S Martinozzi et al ldquo120573-Catenininduces immortalization of melanocytes by suppressingp16INK4a expression and cooperates with N-Ras in melanomadevelopmentrdquo Genes and Development vol 21 no 22 pp2923ndash2935 2007

[217] C Haqq M Nosrati D Sudilovsky et al ldquoThe gene expres-sion signatures of melanoma progressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 102 no 17 pp 6092ndash6097 2005

[218] S Kuphal S Lodermeyer F Bataille M Schuierer B H Hoangand A K Bosserhoff ldquoExpression of Dickkopf genes is stronglyreduced in malignant melanomardquoOncogene vol 25 no 36 pp5027ndash5036 2006

[219] Y-C Lin L You Z Xu et al ldquoWnt inhibitory factor-1 genetransfer inhibits melanoma cell growthrdquoHuman GeneTherapyvol 18 no 4 pp 379ndash386 2007

[220] A M Mikheev S A Mikheeva R Rostomily and H ZarblldquoDickkopf-1 activates cell death in MDA-MB435 melanomacellsrdquo Biochemical and Biophysical Research Communicationsvol 352 no 3 pp 675ndash680 2007

[221] A J Chien E CMoore A S Lonsdorf et al ldquoActivatedWnt120573-catenin signaling in melanoma is associated with decreasedproliferation in patient tumors and amurinemelanomamodelrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 4 pp 1193ndash1198 2009

[222] K SHoekN C Schlegel P Brafford et al ldquoMetastatic potentialof melanomas defined by specific gene expression profiles withno BRAF signaturerdquo Pigment Cell Research vol 19 no 4 pp290ndash302 2006

[223] T L Biechele R M Kulikauskas R A Toroni et al ldquoWnt120573-catenin signaling and AXIN1 regulate apoptosis triggeredby inhibition of the mutant kinase BRAFV600E in humanmelanomardquo Science Signaling vol 5 no 206 p ra3 2012

[224] S K Dissanayake M Wade C E Johnson et al ldquoTheWnt5Aprotein kinase C pathway mediates motility in mela-noma cells via the inhibition of metastasis suppressors andinitiation of an epithelial to mesenchymal transitionrdquo Journalof Biological Chemistry vol 282 no 23 pp 17259ndash17271 2007

[225] A T Weeraratna Y Jiang G Hostetter et al ldquoWnt5a sig-naling directly affects cell motility and invasion of metastaticmelanomardquo Cancer Cell vol 1 no 3 pp 279ndash288 2002

[226] K Burridge and K Wennerberg ldquoRho and RAC take centerstagerdquo Cell vol 116 no 2 pp 167ndash179 2004

[227] X R Bustelo V Sauzeau and I M Berenjeno ldquoGTP-bindingproteins of the RhoRac family regulation effectors and func-tions in vivordquo BioEssays vol 29 no 4 pp 356ndash370 2007

[228] L M Machesky and O J Sansom ldquoRac1 in the driverrsquos seat formelanomardquo Pigment Cell amp Melanoma Research vol 25 no 6pp 762ndash764 2012

[229] M J Davis B H Ha E C Holman R Halaban J Schlessingerand T J Boggon ldquoRAC1P29S is a spontaneously activat-ing cancer-associated GTPaserdquo Proceedings of the NationalAcademy of Sciences of the United States of Amrica vol 110 no3 pp 912ndash917 2013

[230] C R Lindsay S Lawn A D Campbell et al ldquoP-Rex1 is requiredfor efficient melanoblast migration and melanoma metastasisrdquoNature Communications vol 2 p 555 2011

[231] A Li Y Ma M Jin et al ldquoActivated mutant NRas(Q61K)drives aberrantmelanocyte signaling survival and invasivenessvia a Rac1-dependent mechanismrdquo Journal of InvestigativeDermatology vol 132 no 11 pp 2610ndash2621 2012

[232] S Wilhelm C Carter M Lynch et al ldquoDiscovery and develop-ment of sorafenib a multikinase inhibitor for treating cancerrdquoNature ReviewsDrugDiscovery vol 5 no 10 pp 835ndash844 2006

[233] P B Chapman A Hauschild C Robert et al ldquoImprovedsurvival with vemurafenib in melanoma with BRAF V600Emutationrdquo New England Journal of Medicine vol 364 no 26pp 2507ndash2516 2011

[234] R J Sullivan and K T Flaherty ldquoResistance to BRAF-targetedtherapy in melanomardquo European Journal of Cancer vol 49 no6 pp 1297ndash1304 2013

[235] R Anforth P Fernandez-Penas and G V Long ldquoCutaneoustoxicities of RAF inhibitorsrdquo The Lancet Oncology vol 14 no1 pp e11ndashe18 2013

[236] P I Poulikakos C Zhang G Bollag K M Shokat and NRosen ldquoRAF inhibitors transactivate RAF dimers and ERKsignalling in cells with wild-type BRAFrdquo Nature vol 464 no7287 pp 427ndash430 2010

[237] A Hauschild J J Grob L V Demidov et al ldquoDabrafenibin BRAF-mutated metastatic melanoma a multicentre open-label phase 3 randomised controlled trialrdquoThe Lancet vol 380no 9839 pp 358ndash365 2012

[238] P A Ascierto D Schadendorf C Berking et al ldquoMEK162 forpatients with advancedmelanoma harbouring NRAS or Val600BRAFmutations a non-randomised open-label phase 2 studyrdquoThe Lancet Oncology vol 14 no 3 pp 249ndash256 2013

[239] K T Flaherty C Robert P Hersey et al ldquoImproved survivalwith MEK inhibition in BRAF-mutated melanomardquo New Eng-land Journal of Medicine vol 367 no 2 pp 107ndash114 2012

[240] J M Kirkwood L Bastholt C Robert et al ldquoPhase II open-label randomized trial of the MEK12 inhibitor selumetinib asmonotherapy versus temozolomide in patients with advancedmelanomardquo Clinical Cancer Research vol 18 no 2 pp 555ndash5672012

[241] J R Todd T M Becker R F Kefford and H Rizos ldquoSecondaryc-Kit mutations confer acquired resistance to RTK inhibitorsin c-Kit mutant melanoma cellsrdquo Pigment Cell amp MelanomaResearch vol 26 no 4 pp 518ndash526 2013

[242] X Jiang J Zhou N K Yuen et al ldquoImatinib targeting of KIT-mutant oncoprotein in melanomardquo Clinical Cancer Researchvol 14 no 23 pp 7726ndash7732 2008

[243] S Ugurel R Hildenbrand A Zimpfer et al ldquoLack of clinicalefficacy of imatinib in metastatic melanomardquo British Journal ofCancer vol 92 no 8 pp 1398ndash1405 2005

[244] K Wyman M B Atkins V Prieto et al ldquoMulticenter phase IItrial of high-dose imatinib mesylate in metastatic melanomasignificant toxicity with no clinical efficacyrdquoCancer vol 106 no9 pp 2005ndash2011 2006

[245] F S Hodi P Friedlander C L Corless et al ldquoMajor responseto imatinib mesylate in KIT-mutated melanomardquo Journal ofClinical Oncology vol 26 no 12 pp 2046ndash2051 2008

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

22 Scientifica

[246] J Lutzky J Bauer and B C Bastian ldquoDose-dependent com-plete response to imatinib of a metastatic mucosal melanomawith a K642E KIT mutationrdquo Pigment Cell amp MelanomaResearch vol 21 no 4 pp 492ndash493 2008

[247] F S Hodi C L Corless A Giobbie-Hurder et al ldquoImatinib formelanomas harboring mutationally activated or amplified KITarising on mucosal acral and chronically sun-damaged skinrdquoJournal of Clinical Oncology vol 31 no 26 pp 3182ndash3190 2013

[248] D A Oble R Loewe P Yu and M C Mihm Jr ldquoFocus onTILs prognostic significance of tumor infiltrating lymphocytesin human melanomardquo Cancer Immunity vol 9 article 3 2009

[249] D T Alexandrescu T E Ichim N H Riordan et al ldquoImmuno-therapy for melanoma current status and perspectivesrdquo Journalof Immunotherapy vol 33 no 6 pp 570ndash590 2010

[250] T Iida H Ohno C Nakaseko et al ldquoRegulation of cell surfaceexpression of CTLA-4 by secretion of CTLA-4-containing lyso-somes upon activation of CD4+T cellsrdquo Journal of Immunologyvol 165 no 9 pp 5062ndash5068 2000

[251] V D E Van Den Eertwegh ldquoImproved survival with ipili-mumab in patients with metastatic melanomardquo New EnglandJournal of Medicine vol 363 no 8 pp 711ndash723 2010

[252] C Robert L Thomas I Bondarenko et al ldquoIpilimumab plusdacarbazine for previously untreated metastatic melanomardquoNew England Journal of Medicine vol 364 no 26 pp 2517ndash2526 2011

[253] O Hamid ldquoSafety and tumor responses with lambrolizumab(anti-PD-1) in melanomardquo New England Journal of Medicinevol 369 no 2 pp 134ndash144 2013

[254] J D Wolchok ldquoNivolumab plus ipilimumab in advancedmelanomardquo New England Journal of Medicine vol 369 no 2pp 122ndash133 2013

[255] E K Nishimura S R Granter and D E Fisher ldquoMechanismsof hair graying incomplete melanocyte stem cell maintenancein the nicherdquo Science vol 307 no 5710 pp 720ndash724 2005

[256] M L Harris et al ldquoA dual role for SOX10 in the maintenanceof the postnatal melanocyte lineage and the differentiation ofmelanocyte stem cell progenitorsrdquo PLOS Genetics vol 9 no 7Article ID e1003644 2013

[257] N Li M Fukunaga-Kalabis H Yu et al ldquoHuman dermalstem cells differentiate into functional epidermal melanocytesrdquoJournal of Cell Science vol 123 no 6 pp 853ndash860 2010

[258] D Fang T K Nguyen K Leishear et al ldquoA tumorigenicsubpopulation with stem cell properties in melanomasrdquo CancerResearch vol 65 no 20 pp 9328ndash9337 2005

[259] EMonzani F Facchetti E Galmozzi et al ldquoMelanoma containsCD133 and ABCG2 positive cells with enhanced tumourigenicpotentialrdquo European Journal of Cancer vol 43 no 5 pp 935ndash946 2007

[260] J Dou M Pan P Wen et al ldquoIsolation and identificationof cancer stem-like cells from murine melanoma cell linesrdquoCellular amp Molecular Immunology vol 4 no 6 pp 467ndash4722007

[261] A D Boiko O V Razorenova M Van De Rijn et al ldquoHumanmelanoma-initiating cells express neural crest nerve growthfactor receptor CD271rdquo Nature vol 466 no 7302 pp 133ndash1372010

[262] T Schatton G F Murphy N Y Frank et al ldquoIdentification ofcells initiating human melanomasrdquo Nature vol 451 no 7176pp 345ndash349 2008

[263] Y Luo K Dallaglio Y Chen et al ldquoALDH1A isozymes aremarkers of humanmelanoma stem cells and potential therapeu-tic targetsrdquo Stem Cells vol 30 no 10 pp 2100ndash2113 2012

[264] A Roesch M Fukunaga-Kalabis E C Schmidt et al ldquoAtemporarily distinct subpopulation of slow-cycling melanomacells is required for continuous tumor growthrdquo Cell vol 141 no4 pp 583ndash594 2010

[265] A Roesch A Vultur I Bogeski et al ldquoOvercoming intrinsicmultidrug resistance in melanoma by blocking the mitochon-drial respiratory chain of slow-cycling JARID1B(high) cellsrdquoCancer Cell vol 23 no 6 pp 811ndash825 2013

[266] I Vsquez-Moctezuma M A Meraz-Ros C G Villanueva-Lpez et al ldquoATP-binding cassette transporter ABCB5 geneis expressed with variability in malignant melanomardquo ActasDermo-Sifiliograficas vol 101 no 4 pp 341ndash348 2010

[267] N Y Frank A Margaryan Y Huang et al ldquoABCB5-mediateddoxorubicin transport and chemoresistance in human malig-nantmelanomardquoCancer Research vol 65 no 10 pp 4320ndash43332005

[268] H T Khong Q J Wang and S A Rosenberg ldquoIdentification ofmultiple antigens recognized by tumor-infiltrating lymphocytesfrom a single patient tumor escape by antigen loss and loss ofMHC expressionrdquo Journal of Immunotherapy vol 27 no 3 pp184ndash190 2004

[269] J Du A J Miller H R Widlund M A HorstmannS Ramaswamy and D E Fisher ldquoMLANAMART1 andSILVPMEL17GP100 are transcriptionally regulated by MITFin melanocytes and melanomardquo American Journal of Pathologyvol 163 no 1 pp 333ndash343 2003

[270] T AManolio F S Collins N J Cox et al ldquoFinding themissingheritability of complex diseasesrdquo Nature vol 461 no 7265 pp747ndash753 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom