review article cellular signaling pathway...

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 803171 16 pageshttpdxdoiorg1011552013803171

Review ArticleCellular Signaling Pathway Alterations and Potential TargetedTherapies for Medullary Thyroid Carcinoma

Serena Giunti1 Alessandro Antonelli2 Andrea Amorosi1 and Libero Santarpia3

1 Department of Pathology Centro Oncologico Fiorentino Sesto Fiorentino 50019 Firenze Italy2 Department of Internal Medicine University of Pisa School of Medicine 56100 Pisa Italy3 Translational Research Unit Department of Oncology Istituto Toscano Tumori 59100 Prato Italy

Correspondence should be addressed to Libero Santarpia lsantarpiausl4toscanait

Received 12 November 2012 Revised 8 January 2013 Accepted 10 January 2013

Academic Editor Eleonore Frohlich

Copyright copy 2013 Serena Giunti et al 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

Parafollicular C-cell-derived medullary thyroid cancer (MTC) comprises 3 to 4 of all thyroid cancers While cytotoxictreatments have been shown to have limited efficacy targeted molecular therapies that inhibit rearranged during transfection(RET) and other tyrosine kinase receptors that are mainly involved in angiogenesis have shown great promise in the treatmentof metastatic or locally advanced MTC Multi-tyrosine kinase inhibitors such as vandetanib which is already approved for thetreatment of progressive MTC and cabozantinib have shown distinct advantages with regard to rates of disease response andcontrol However these types of tyrosine kinase inhibitor compounds are able to concurrently block several types of targets whichlimits the understanding of RET as a specific target Moreover important resistances to tyrosine kinase inhibitors can occur whichlimit the long-term efficacy of these treatmentsDeregulated cellular signaling pathways and genetic alterations inMTC particularlythe activation of theRASmammalian target of rapamycin (mTOR) cascades andRET crosstalk signaling are now emerging as noveland potentially promising therapeutic treatments for aggressive MTC

1 Introduction

Medullary thyroid carcinoma (MTC) is a rare neuroen-docrine cancer that originates from thyroid parafollicular cal-citonin-(CT-) producing cells MTC accounts for approxi-mately 4 of all thyroid malignancies approximately 75 ofthese cases occur in the sporadic form and 25 occur inthe hereditary form [1ndash3] MTC usually has a favorableprognosis with a 10-year survival rate of 70ndash80 if itis diagnosed and treated at an early stage when the tumoris confined to the thyroid [4] Unfortunately most casesof MTC present at diagnosis with metastases to the localand regional lymph nodes and to distant organs especiallythe lungs liver and bones [5] Patients with metastaticMTC have a 10-year over-all survival rate of 40 andmetastasis is the main cause of death in patients with MTC[4 6] Locally advanced and distant metastatic diseases areincurable as surgical resection and conventional radio- andcytotoxic chemotherapies are not effective against metastaticMTC [7 8] Clinical trials of various combinations of

chemotherapeutic drugs have yielded unsatisfactory results[9 10] However research over the last years has led toa good understanding of the genetic defects and alteredmolecular pathways that are associated with the developmentof MTC Thus multiple promising therapeutic agents thattarget these genetic alterations have been developed totreat progressive and advanced MTC Activating mutationsof the tyrosine kinase receptor (TKR) rearranged duringtransfection (RET) are believed to be the primary oncogenicevent in a majority of MTC cases This discovery has ledto the development and introduction of targeted therapiessuch as tyrosine kinase inhibitors (TKIs) that target RETSeveral TKIs directed toward RET kinase have been tested invitro preclinical and clinical studies with promising resultsUnfortunately these agents are not likely to be curative asthe longest duration of response observed was approximately4 years and the maintenance of agent-dependent effectsmay require continuous therapies [7] which are not withoutimportant side effects The main reasons for the failure ofthese agents to cure MTC are the development of resistance

2 International Journal of Endocrinology

to TKIs that target the RET and other cell receptors andthe activities of other signal transduction pathways that areinvolved in MTC tumorigenesis and progression but notdirectly targeted by TKIs In recent years the discovery ofmechanisms of resistance to TKIs and of several other mole-cular events that contribute to MTC transformation andmetastasis suggested that combinatory therapy may result ina more significant tumor growth inhibition This has led tothe development of novel compounds that have been used inseveral clinical trials including TKIs that can target multipleTKRs simultaneously in addition to RET and agents that cantarget other altered signaling pathways Other studies havedemonstrated the potential for immunotherapy in combi-nation with agents that target signal transduction pathwaysthat are important for MTC growth [5] Because the aim ofthese targeted therapies is to extend lifespan and increasethe quality of life it is very important to limit the toxicitiesof therapeutic agents either alone or in combination Thepossibility of testing these novel drugs in vitro (in primarythyroid cancer cells) and in vivo may help to improve thepersonalization of treatments [11]

2 Key Cellular Signaling Pathways andAlterations in MTC

21 RET Pathway The role of the RET oncogene in thetumorigenesis of MTC has been characterized extensively[12] The RET gene encodes a transmembrane tyrosinekinase that binds to glial cell line-derived neurotrophicfactor (GDNF) family ligands [13] RET signaling leads tothe activation of the RASmitogen-activated protein kinase(MAPK) and the phosphatidylinositol 31015840 kinase (PI3K)Aktpathways and has key roles in cell growth differentiationand survival Activating point mutations of the TKR REThave been reported in nearly all hereditary cases of MTCsome of these mutations are included in theMEN2A familialMTC or MEN2B syndromes in which there is a geno-typicphenotypic correlation between the type of RET muta-tion and clinical features RET mutations are also found in30ndash50 of sporadicMTCs Germlinemutations in the RETproto-oncogene are responsible for hereditary MTC whilesomatic RET mutations are responsible for sporadic MTC[14] These data provide a strong rationale for targeting RETin selective cancer therapy However this paper will mainlyfocus on additional cellular signaling pathways other thanRET responsible of MTC tumorigenesis and progression andpotential targeted approaches for the treatment of advancedor metastatic MTC

22 Additional Signaling PathwaysThat AccelerateMTC Prog-ression Although activating mutations of the TKR RET arebelieved to be the primary oncogenic event in the deve-lopment of amajority ofMTC cases it is clear that RET coop-erates with other signal transduction pathways to promoteMTC tumorigenesis

221 Tyrosine Kinase Receptors other than RETAre Implicatedin MTC Tumorigenesis In addition to RET other kinase

receptors may play a role in the development and progressionof MTCs [15]

Similar to the RET receptor the epidermal growth factorreceptor (EGFR) is a TKR that is associated with the regula-tion of cell growth proliferation and apoptosis Dimerizationof the receptor following ligand binding results in transphos-phorylation and the subsequent activation of several down-stream signal pathways EGFR has been shown to be fre-quently overexpressed in various types of thyroid carcinomasincluding MTC and to play a role in cancer developmentand progression [16] In contrast a recent report analyzingdifferent MTC on tissue microarrays has demonstrated thatonly 20 of cases revealed moderate to strong reactivity forEGFR whereas the majority of the cases revealed weak andvery focal positivity [17] A different study has shown thatEGFR-activating mutations are rare in MTC With respect tothe numbers of EGFR gene copies in MTCs the researchersdid not detect amplifications but did find polysomes in 15 ofthe examined tumors [18] Additionally EGFR was activatedin a subset of MTCs which suggests that this subset ofpatients might benefit from drugs that target also EGFRRecent findings have shown that the ligand-induced activa-tion of EGFR can stimulate RET activation beyond signalingand growth stimulation [19] Several EGFR inhibitors havebeen shown to markedly inhibit the growth of the MTC TTand MZ-CRC-1 cell lines Because RET activation seems tobe influenced by EGFR a recent study investigated whetherEGFR activation could be related to specific RET mutationsin MTCs The researchers found that tumors with the mostaggressive RET mutations (in codons 883918) exhibitedreduced EGFR expression compared to otherRETmutationsIt could be speculated that themost aggressiveRETmutationsare less dependent on EGFR activation [18] In fact in thework by Croyle et al [19] in which cell lines with RETmutations in codon 634 and codon 918 were compared theeffect of EGFR inhibition on the codon 918 mutated cell lineappeared to be reduced in agreement with the previous dataBecause the activation status of EGFR seems to be relatedto RET activation EGFR activation has been examined inRET-negative tumors [18] However no differences havebeen found in EGFR activation between RET-positive and-negative tumors which likely indicates that other molecularmechanisms lead to RET activation such as increased RETgene copy numbers altered promoter activity or increasedtranscription in the RET mutation-negative tumors Thesedata suggest that EGFR status determination in MTCs mightbe important but certainly deserves further investigations

The vascular endothelial growth factor receptor (VEGFR)pathway is also important in the pathogenesis of MTCThereare three transmembrane receptors that mediate the angio-genic and lymphogenic effects ofVEGFVEGFR-1 VEGFR-2and VEGFR-3 VEGFR-2 is thought to be implicated pri-marily in tumor growth and metastasis Overexpression ofVEGF and VEGFR-2 has been found in MTC compared tonormal thyroid tissue [20] The VEGF proteins (VEGF-A BC and D) which are secreted by tumor cells act as ligandsfor theVEGFR-2 receptors on endothelial cells and promote asignaling cascade through different pathways such as PLC-120574-PKC-Raf-MEK-MAPK and PI3K-Akt that stimulate cellular

International Journal of Endocrinology 3

proliferation migration and survival and induce neoangio-genesis [21] Angiogenesis is an essential alteration in cellphysiology that predisposes the development of malignancyand is fundamental in tumor growth and metastasis [22]Similarly to EGFR the overexpression of VEGFR-2 in MTChas been shown to correlate with metastasis [18]

Several multitargeting tyrosine kinase inhibitors thatblockVEGFRhave shownpromising clinical antitumor activ-ity unfortunately inmost thyroid carcinomas and other solidtumors the antiangiogenic effects are often only transitoryand really often may have late paradoxically protumorigeniceffects Additionally it seems that a modest significant asso-ciation has been observed between VEGFR-2 expression andRET mutation status in primary tumors [18]

The MET proto-oncogene codes for the TK receptor forthe hepatocyte growth factor (HGF)TheHGF-MET interac-tion activates signaling pathways that mediate cell adhesionandmotility MET hyperactivation reportedly correlates withthe metastatic abilities of tumor cells [23] MET and HGFcoexpression has been observed in a subset of MTC tumorsand is associated with multifocality in MTC [24] whichmakes this interaction a potentially important target In onereport mutations in the MET RTK were detected in MTC[25] Importantly RET can induce the overexpression of c-MET in this type of thyroid tumor [26]

The fibroblast growth factor receptor 4 (FGFR4) has alsobeen reported to be overexpressed in MTC cell lines Inhibi-tion of FGFR phosphorylation with the small molecule FGFRinhibitor PD173074 resulted in an arrest of cell proliferationand tumor growth [27] Moreover the dual inhibition of RETand FGFR combined with tyrosine kinase inhibitors resultedin greater suppressions of cell proliferation in vitro and tumorcontrol in vivo than that whichwas achievedwith either agentalone These data highlight RET and FGFR4 as therapeutictargets and suggest a potential role for the use of combinedtyrosine kinase inhibitors in the management of inoperablemedullary thyroid cancers [28]

Finally the platelet-derived growth factor receptor(PDGFR) also seems to play a role in differentiated thyroidcancer [29] although its role and function have not beenfully investigated in MTC

222 Other Signaling Pathways That Contribute to MTCTumorigenesis Several other signal transduction pathwayshave been implicated as contributors to MTC tumor growthas illustrated in two recent studies [5 30] These pathwaysinclude RET interactions with pRB p53 p18 and p27 aswell as the phosphatidylinositol 3-kinaseAKTmTOR andRasRafMEKERK pathways

RET Interactions with Tumor Suppressor Genes

(a) pRB and p53The tumor suppressor genesRB1 (retinoblas-toma pRB protein) and TP53 (p53 protein) are frequentlymutated in human cancers and it appears that in cancerboth pathways must be inactivated to overcome senescenceor apoptosis There is extensive genetic evidence that thepRB and p53 pathways are involved in MTC in rodents Forexample RB1-deficient mice developed MTC [31] Further

the loss of TP53 further increased MTC formation in RB1-deficient mice [32 33] It has been shown that in mice RETcooperates with the inactivation of pRBp53 to cause experi-mental MTC pRB+minusp53+minus mutant mice have been shownto acquire RET mutations that are analogous to activatinggermline mutations that are observed in humanMEN2A andfamilial medullary thyroid carcinoma (FMTC)This suggeststhat murine MTC requires mutational dysregulations withinboth the RET and nuclear tumor suppressor gene pathways[34] However mousemodelsmay notmimic human diseaseand a systematic analysis of the genes in the RB1 and TP53pathways in human samples will help to clarify their roles inhuman MTC formation This information may be importantfor the development of novel targeted therapeutic approachesfor MTC

(b) p18 and p27 Thyroid tumors show low expression of thecyclin-dependent kinase inhibitor (CDKI) p27 (Kip1) andrecent evidence demonstrates that p27 is downregulated bythe active RET mutant RETPTC1 which is found in papil-lary thyroid carcinomas These data implicate decreased p27activity as an important event during thyroid tumorigenesisHowever p27minusminus mice develop MEN-like tumors only incombination with the loss of p18 (Ink4c) another CDKIThissuggests that p18 and p27 are functional collaborators in thesuppression of tumorigenesis that the loss of both is criticalto the development of MEN tumors and that both p18 andp27 are regulated by RET [35]

PI3K-AKT-mTOR Pathway The PI3K-AKT-mammalian tar-get of the rapamycin (mTOR) cascade is important in tumori-genesis due to its ability to promote cell growth proliferationand survival Several examples provide evidence to supporta role for the activation of the PI3KAKTmTOR signalingcascade in medullary thyroid cancer [36ndash38]

Several mechanisms have been shown to be involvedin the activation of PI3K signaling in medullary thyroidcancer

A mutation of MEN2A (RET-MEN2A) has been shownto activate PI3K and its downstream effector the serinethreonine kinase AKTprotein kinase B [35] Previous studieshave demonstrated that a mutation of Tyr-1062 which isthe intracellular docking site for Shc and Enigma on RETabolishes the RET-MEN2A transforming activity [39] Thesestudies further revealed that the mutation of Tyr-1062 abro-gates the binding of the p85 regulatory subunit of PI3Kto RET-MEN2A and subsequent stimulation of the PI3KAKT pathway Furthermore retroviral transduction of ratfibroblasts with a dominant-interfering form of PI3K wasshown to suppress RET-MEN2A-dependent transformationwhereas the overexpression of AKT enhanced RET-MEN2Aoncogenic potential In summary these data are consistentwith the notion that RET-mediated cell-transforming effectsare critically dependent on the activation of the PI3KAKTmTOR pathway [36]

In cell lines the PI3K-AKTmTOR pathway has beenshown to be important in the pathogenesis of MEN2B[40] RET-MEN2B (RET M918T) is more effectively auto-phosphorylated at RET Y1062 than is RET-MEN2A which


subsequently leads to increased constitutive activation ofthe Rasmitogen-activated protein kinase (MAPK) and PI3KAKTmTOR signaling cascades [41]

Furthermore previous data report other possible mech-anisms of PI3K activation in thyroid carcinomas such as theoverexpression of RAI (ShcCN-Shc) which is a substrate ofRET oncoproteins [5 42]

Finally the loss of expression of the phosphatase andtensin homologue (PTEN) gene a tumor suppressor genemay have a role in the activation of PI3KAKT in thyroidtumors The absence of functional PTEN protein expressionhas been observed in various cancer cells and has led tothe constitutive activation of downstream components of thePI3K pathway including the Akt and mTOR kinases Pre-clinical models showed that inactivation of these kinases isable to reverse the effects of PTEN loss [43] These data raisethe possibility that drugs that target either these kinases orPI3K itself might have significant therapeutic activity againstPTEN-null cancers

Mutations in major nodes of this signaling cascade havebeen observed in human cancers these mutations includegain-of-function mutations and amplifications of the genesencoding PI3K and AKT [44 45] No systematic geneticanalysis of PI3K pathway components has been reported inMTC However a recent study on a series of 49 MTCs hasshown that the PI3K genes were not mutated and that theactivation of the PI3K pathway was significantly associatedwith the status of RET mutations [46] In fact using proteinexpression analysis the same authors confirmed that theAKTmTORpathwaywas highly activated inMTC especiallyin cases with germline RET mutations Interestingly thisassociation was not dependent on the type of mutation (ineither the codons of the juxta-membrane or of the tyrosinekinase portion of the receptors) but was dependent on thehereditary nature of the mutation In contrast medullarycarcinomas with sporadic RET mutations or with wild-type RET were observed with heterogeneous expression ofAKTmTOR pathway molecules which suggests the need forfurther elucidation of alternative activationmechanisms [46]In a recent study the PI3KAKTmTOR pathway was shownto be activated in MTC particularly in the metastatic lymphnodes and this pathway was shown to sustain malignantfeatures of different MTC cell models [47] Moreover ithas been shown that the selective inhibition of the mTORpathway in a germline-RET-mutated MTC cell line caneffectively decrease cell viability and block the phosphory-lated status of mTOR signaling molecules which confirmspreviously published in vitro data [48] Another study usedmetformin which is an antidiabetic agent that decreases theproliferation of cancer cells through the 51015840-AMP-activatedprotein kinase-dependent inhibition of mTOR in MTCcell lines to show that the growth-inhibitory effects on thecells were associated with the downregulation of both themTOR6SK and pERK signaling pathways [49] Altogetherthese results strongly suggest that mTOR might be a veryefficacious target in patients with advanced or metastaticMTC

Noteworthy mTOR inhibitors are currently being usedin clinical trials for the treatment of medullary thyroid car-cinoma

Ras-Raf-MEK-ERK Pathway Unlike hereditary MTC inwhich RET mutations are the critical events in sporadicMTC the genetic or molecular biomarkers have not beenfully established Ras is a frequently mutated oncogene in abroad spectrum of human tumors including thyroid carci-noma mainly in the follicular forms [50] In this contextinvestigators aimed to determine whether mutations in theRas oncogene could play a possible role in the carcinogenesisof sporadic MTC The initial study analyzed 15 sporadicMTCs for mutations in known hot spots (codons 12 13 and61) of the H- and K-Ras oncogenes by a direct sequencingtechnique although no Ras mutations were detected in anyof the examined tumors [50] A different study on 49 MTCconfirmed that Ras mutations are a rare event in this typeof tumor regardless of RET mutational status [46] By con-trast different studies have demonstrated the presence ofRas mutations in MTC A mutational screening for H- K-N-RAS and BRAF in 13 sporadic and inherited MTCsrevealed one sporadic RET-negative MTC (stage III) witha mutation in the H-Ras codon 13 (G13R) [51] Anotherstudy analyzed 188 hereditary and sporadic MTCs for Rasmutational status revealing a low prevalence of mutationswhich were confined only to RET-negative sporadic MTC[52] Furthermore recent studies have reported a quite highprevalence of Ras mutations in sporadic MTC particularlyin RET-negative MTC 68 (17 of 25) of the RET-negativeMTCs had mutations of Ras compared to only 25 (140) ofthe RET-positive MTCs [53] These findings were confirmedby a different study that analyzed 17 sporadicMTCs by exomesequencing and found dominant and mutually exclusiveoncogenic mutations in RET and RAS (H- and K-Ras) genesin 17 sporadic MTCs [54] As expected most Ras mutationscorresponded to mutational hot spots in exons 2 and 3although some mutations were also detected in exon 4 Thisstudy confirms that RET and Ras mutations are mutuallyexclusive and that they are probably 2 different oncogenicdriver events in MTC [55] These results suggest that theactivation of the proto-oncogenes Ras and RET representsalternative genetic events in sporadic MTC tumorigenesisand that more sensitive sequencing techniques such as nextgeneration sequencing are necessary to detect mutations Fordecades the Ras and Raf families of oncogenes have beenknown to be transforming genes However in many normalcultured cell types the sustained expression of activated Rasor its downstream effector Raf can elicit cell cycle arrestor senescence The RasRaf-mediated growth arrest has beenproposed as a defense mechanism against the inappropriateactivation of the RasRaf signal transduction pathway [5657] According to this hypothesis spontaneous mutationsin Ras genes which may occur stochastically in all celltypes would be innocuous for the organism because thesemutations would lead to growth arrest or senescence Hencefor carcinogenesis to occur in response to RasRaf activationthe growth arrest response must be inactivated Thus cell


transformation may involve additional events The growtharrest response to RasRaf activation is not limited to normalcells Several tumor cell lines that were derived from differenttumor types including medullary thyroid carcinoma alsoexperienced growth arrest usually accompanied by differ-entiation or senescence [58ndash60] These findings indicatethat some tumors retain a capability for growth arrest inresponse to RasRaf activation The mechanism by whichRas or Raf activation can induce growth arrest is not com-pletely understood Some investigators have reported thatRas or Raf activation could induce the expression andsecretion of a protein that mediated differentiation and G1cell cycle arrest in MTC cells By protein purification andmass spectrometry this protein has been identified as theleukemia inhibitory factor (LIF) STAT3 activation was nec-essary for LIF-mediated growth arrest and differentiationin MTC cells In addition the RasRafMEKERK pathwaycould also mediate growth arrest and differentiation by asecond mechanism that is independent of LIFJAKSTAT3This novel autocrine-paracrine mechanism which mediatescrosstalk between the RasRafMEKERK and the JAK-STATpathways defines a novelmechanism of RasRaf-induced cellgrowth arrest [61 62]

Raf-1 activation in human MTC TT cells resulted in thephosphorylation of GSK-3beta The inactivation of GSK-3beta in TT cells by well-known GSK-3beta inhibitors suchas lithium chloride (LiCl) and SB216763 is associated withboth growth suppression and a significant decrease in neu-roendocrinemarkers such as human achaete-scute complex-like 1 and chromogranin A Growth inhibition by GSK-3beta inactivation was found to be associated with cell cyclearrest due to an increase in the levels of cyclin-dependentkinase inhibitors such as p21 p27 and p15 AdditionallyTT xenografts mice treated with LiCl showed a signifi-cant reduction in tumor volume compared with those thatwere treated with a control Therefore GSK-3beta is a keydownstream target of the Raf-1 pathway in TT cells and theinactivation of GSK-3beta alone is sufficient to inhibit thegrowth of TT cells both in vitro and in vivo [63]

120573-Catenin Pathway 120573-Catenin is a ubiquitously expressedmultifunctional protein that plays an important role incellular adhesion A novel RET-120573-Catenin signaling pathwaywas found to be a critical contributor to enhanced cell pro-liferation and tumor progression in thyroid cancer Gujralet al showed that RET could induce 120573-Catenin-mediatedtranscription cell proliferation and transformation in vitroand that 120573-Catenin nuclear localization and subsequentmediation of 120573-Catenin by RET are key secondary eventsin tumor growth and metastasis in vivo [64] This novelinteraction suggests a mechanism that may underlie thebroad and early metastatic potential of MTCThese data sug-gest an unrecognized role for 120573-Catenin signaling that mayhave implications for tyrosine kinase-mediated tumorigen-esis in multiple tumor types and provide another potentialtarget for therapeutic agents In support a recent study per-formed on tissue microarray observed that WNT pathway

proteins including Wisp-1 Wisp-2 and 120573-Catenin wereactively expressed in MTC [17]

NF-120581B Pathway The nuclear factor kappa-B (NF-120581B) pro-teins a family of transcription factors that are found virtuallyin all cells are known to play crucial roles in the growth ofmany human malignancies The ability of NF-120581B to targeta large number of genes that regulate cell proliferationdifferentiation survival and apoptosis provides clues towardsits dysregulation during the processes of tumorigenesismetastatic progression and therapeutic resistance of tumorsNF-120581B is constitutively active in MTC through the RET-induced phosphorylation ubiquitination and proteosomaldegradation of inhibitors of NF-120581B (IkB) which allows NF-120581B to enter the nucleus and bind to DNA [65] NF-120581B isfrequently activated in MTC and the activation of RET bysomatic or germline mutations may be responsible for NF-120581B activation in these tumors [5] These results suggest thatthe NF-120581B pathway may be an important target for drugdevelopment in MTC

Novel Protein Targets A recent study examined the expres-sion of proteins involved in angiogenesis inflammationapoptosis cell cycle cell-to-cell contact and carcinogenesisusing high-throughput tissue microarrays from 23 patientswith MTC These authors identified several novel potentiallyimportant protein targets such as COX-12 Bcl-2a Gst-120587Gli-1 Gli-2 Gli-3 and Bmi-1 that may be therapeuticallytargeted in MTC For example COX-1 and COX-2 whichare two inflammation-related factors were significantlyexpressed in these cases suggesting that nonsteroidal anti-inflammatory drugs may provide benefit in some patientswith MTC Then the finding of antiapoptotic Bcl-2a andGst-120587 overexpression in MTC suggests that Bcl-2a and Gst-120587 inhibitors might be a treatment option for patients withadvanced or metastatic MTC The same is applied to Gli-1Gli-2 and Gli-3 members of Sonic Hedgehog Homolog(SHH) pathway and Bmi-1 a cell-cycle marker that resultedoverexpressed in theseMTC samples [17]The studies of thesemarkers particularly the members of the SHH may improveour understanding of mechanism of resistance to currentchemotherapeutic andor TKI regimens and identify novelpotential therapeutic approaches

3 Potential Targeted Therapies for MTC

31 Tyrosine Kinase Receptor Inhibitors Due to increasedknowledge of the molecular pathogenesis of MTC thera-peutic agents that target specific altered pathways havebeen developed (Figure 1) Because the alterations of proteinkinases and their pathways are involved in MTC develop-ment several tyrosine kinase receptors inhibitors (TKIs) havebeen tested in vitro preclinical and clinical studies [66] RETis certainly an attractive target for several types of tumorsparticularly for parafollicular C-cells-derived tumors whichare addicted to RET and its activity [66] TKIs are smallorganic compounds that affect tyrosine kinase-dependentoncogenic pathways by competing with ATP-binding sitesof the tyrosine kinase catalytic domains [67] Occupation


MA

PK p

athw

ay

MA

PK p

athw

aySorafenibSunitinibMotesanibImatinibXL184

SorafenibSunitinibMotesanibImatinib

VandetanibSorafenibMotesanib

VandetanibXL184SorafenibSunitibMotesanib

Vandetanib

VandetanibXL184SorafenibSunitinibImatinib

Everolimus

SorafenibCRAF

Transcription factors

Proliferation

Differentiation

Survival GrowthMigration

RETEGFR

MET

FGRFKIT

Thyroid tumor

cell

PTEN

Bortezomib

Endothelial vascular

cell

Endotheliallymphatic

cell

VEGFR3 VEGFR2PDGFR


Proliferation Survival Migration

Downstream cellular effects

Differentiation

IKK

PI3K

mTOR

MEK

RAS

Physiological effects


ERK

BRAF

MEK

ERK

PI3K

AKT AKT

mTOR

BRAF

RASJAK

STAT

NF120581B

Growth tumor

Lymphangiogenesisangiogenesis

Metastasis

Figure 1 This figure schematically shows the tyrosine kinase receptors such as RET EGFR FGFR MET KIT VEGFR PDGFR andsome of their downstream effectors The two most important oncogenic signaling pathways are PI3KAKTmTOR and RasRafMEKERKThe activation of the pathways may transduce different transcription factors and induce tumor and endothelial cell proliferation survivalmigration with subsequent tumor growth lymphangiogenesisangiogenesis and metastasis Moreover the main tyrosine kinase inhibitorsand their targets are shown

of these sites inhibits autophosphorylation and activation ofthe tyrosine kinases and prevents the further activation ofintracellular signaling pathways TKIs can be specific to oneor several homologous tyrosine kinases

Several TKIs that are directed against RET kinase havebeen developed for the treatment of MTC but there is nocurrently available tyrosine kinase inhibitor specific to RETHowever several multitargeted tyrosine kinase inhibitorshave demonstrated significant activity against RET (Table 1)including vandetanib sorafenib motesanib imatinib andsunitinib [66 68] The inhibition of only one kinase receptormay induce the compensatory activation of other TKs andconsequent resistance to treatment with TKIs [69 70]There-fore the simultaneous inhibition of multiple activated TKsmay be the best way to approach MTC [71ndash73] TKIs that arecurrently undergoing testing in clinical trials are described asfollows

Vandetanib (ZD6474) is an oral TKI that targets RETVEGFR-2 and -3 and at higher concentrations EGFR [74]

Vandetanib selectively inhibits pathways that are critical fortumor growth and angiogenesis without leading to directcytotoxic effects on tumor or endothelial cells [75] Most ofthe mutant RET oncoproteins have demonstrated sensitiv-ity to vandetanib while mutations in which valine 804 issubstituted by either leucine or methionine (as observed insome cases ofMEN2A) rendered the RET kinase significantlyresistant to vandetanib This resistance is likely due tosteric hindrance as the Val804Gly mutation increased thesensitivity of RET to vandetanib [76] When RET activitywas inhibited overstimulation of EGFR was able to partiallycompensate for RET through a partial rescue of MAPK path-way activation The inhibition of EGFR by vandetanib hasbeen shown to prevent this rescue of MAPK pathway acti-vation These data support the idea that the dual inhibitionof RET and EGFR is important as it may overcome therisk of an escape by MTC cells from a blockade of RETthrough the compensatory overstimulation of EGFR [69] Inaddition the expression of EGFR has been demonstrated in


Table1Maintargeted

therapiesc

urrentlyin

usefor

thetreatmento

fmedullary

thyroidcancer

Keytargets

httpwwwclinicaltrialsgov

Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Vand

etanib

(ZD6474)

radicradic

radicradic

01

Bortezom

ib

Notyetrecruitin

gNCT

0166117

9(12)

Recruitin

gNCT

005140

46(12)

NCT

01496313

(4)

Activenotrecruitinghas

results

NCT

00410761

(3)

NCT

00358956

(2)

NCT

00098345

(2)

Activenotrecruiting

NCT

000923247(12)

NCT

01298323

(3)

With

draw

nDocetaxel

NCT

000937417

XL184

(Cabozantin

ib)radic

radicradic

radicradic

000

4

Activenotrecruiting

NCT

00704730

(3)

NCT

00215605

(1)

Available

NCT

01683110

MainTK

IsSorafenib

(BAY

-43-9006)radic

radicradic

radicradic

radicradic

00059ndash0

05

Activenotrecruiting

NCT

00390325

(2)

Unknow

nlowastlowast

NCT

006542387(2)

Sunitin

ib(SU11248)

radicradic

radicradic

radic022ndash13

Activenotrecruiting

NCT

00381641

(2)

NCT

00519896

(2)

NCT

005106

40(2)

Motesanib

(AMG-706)

radicradic

radicradic

005ndash0

09

Completed

NCT

00121628

(2)

Imatinib

(STI571)

radicradic

radic5ndash37

XelodaD

acarbazine

Activenotrecruiting

NCT

00354523

(12)


Table1Con

tinued

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Everolim

us(RAD001)

mTO

RPasireotid

ePasireotid

e

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

NCT

01164176

(2)

Unknow

nlowastlowast

NCT

01118

65(2)

Main

Non

-TKIs

Bortezom

ib(PS-341)

proteosome

Vand

etanib

Activenotrecruiting

NCT

00923247

(12)

Pasireotid

e(SOM230)

sst1

35(som

atostatin

receptors)

Everolim

usEv

erolim

us

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

lowastIC

50RE

T[120583M]50

inhibitory

concentration

lowastlowastStatus

hasn

otbeen

verifi

edin

morethantwoyears


tumor-associated endothelial cells [77] In this respect anti-EGFR agents have been shown to block the proliferationand migration of endothelial cells Therefore the anti-EGFRactivity of vandetanib might result in an additional directantiangiogenetic mechanism One phase II clinical trialshowed the antitumor activity of vandetanib (300mgday)in patients with hereditary MTC In this study 20 of thepatients showed a partial response to vandetanib (gt30reduction in tumor diameter) while an additional 53 ofpatients presented with disease stability at 24 weeks Only1 patient showed disease progression while receiving thisagent Of interest this patient was not affected by any oftwo RET mutations correlated to vandetanib resistance inwhich valine 804 is substituted by leucine or methioninebut by Y791F RET germline mutation [78] Another clinicaltrial in which 19 patients with hereditary MTC were treatedwith vandetanib (100mgday) showed similar results [79]Vandetanib is the only TKI approved for the treatment ofsymptomatic or progressive MTC in patients with unre-sectable locally advanced or metastatic disease [80] Theapproval of vandetanib inApril 2011 by theUS Food andDrugAdministration (FDA) was based on the results of the phaseIII clinical trial ldquoStudy D4200C00058rdquo in which 331 patientswith unresectable locally advanced or metastatic MTC wererandomly assigned to receive vandetanib (231) or a placebo(100) This study showed that the median progression-freesurvival duration (PFS) was 11 months longer in the groupthat received vandetanib than in the placebo control groupand that 45 of the patients had an objective response rate(ORR) Common adverse events (any grade) occurred morefrequently with vandetanib compared to the placebo andincluded diarrhea (56 versus 26) rash (45 versus 11)nausea (33 versus 16) hypertension (32 versus 5) andheadache (26 versus 9) [81]The impact of overall survivalof MTC patients treated with this compound is presentlyunknown

XL184 (cabozantinib) is an oral selective inhibitor ofRET c-MET andVEGFR-2 c-MET activation triggers tumorgrowth and angiogenesis A phase I trial has shown clinicalbenefits of XL184 in patients with MTC [82] These resultshave led to the expansion of anMTC-enriched patient cohortThephase I trial results indicated that cabozantinib is active inpatients withMTC including those who harbor somatic RETmutations and are potentially at high risk for progression anddeath [83] A global phase III pivotal study inMTC is ongoing(httpwwwClinicalTrialsgov number NCT00704730)

Sorafenib (BAY 43-9006)is a multikinase inhibitor thattargets BRAF VEGFR-2 VEGFR-3 KIT (a stem cell growthfactor proto-oncogene involved in cell survival and differen-tiation) and PDGFR This drug has been shown to stronglyinhibit RET kinase activity in vitro [84] A phase II clinicaltrial in which sorafenib (400mgtwice daily) was given to21 patients with metastatic medullary carcinoma reportedthat of the patients with sporadic MTC 875 achieved dis-ease stability and 63 demonstrated partial responses Themedian PFS was 179 monthsThe treatment was prematurelyterminated in MTC hereditary patients due to slow accrual[85] In a similar trial all 5 patients treated with sorafenibexhibited partial responses [86] Recently a study of sorafenib

was conducted on advanced thyroid carcinoma patients andpartial responses were reported in six out of the 12 (50)patients with MTC although the small number of patientsrequires further prospective studies [87]

Sunitinib (SU011248) is a small molecule inhibitor thattargets many of the same protein kinases as sorafenib includ-ingVEGFR PDGFR KIT andRET In a phase II clinical trial33 patients with either well-differentiated thyroid carcinomaor MTC were treated with sunitinib One patient had acomplete response 28 of the patients had partial responsesand 46 of the patients exhibited disease stability [88] Theintermediate results of the phase II THYSU study also showedthe efficacy of sunitinib in advanced medullary thyroid car-cinoma The final results are yet to be released [89]

Motesanib (AMG 706) is a multikinase inhibitor that tar-gets VEGFR receptors 1 2 and 3 PDGFR andKIT and exertsantiangiogenic and direct antitumor activities [90] A phaseII study performed in 91 patients with locally advanced ormetastatic progressive or symptomatic MTC demonstratedthat although the objective response ratewas low a significantproportion of theMTCpatients (81) achieved disease stabil-itywhile receivingmotesanib [91] A recent study investigatedthe effects ofmotesanib onwild-type andmutant RET activityin vitro and on tumor xenograft growth in a mouse model ofMTC The results of this study demonstrated that motesanibinhibited thyroid tumor xenograft growth predominantlythrough the inhibition of angiogenesis and possibly via thedirect inhibition of VEGFR2 and RET which were expressedin tumor cells These data suggest that angiogenic pathwaysand specifically the VEGF pathway are still important forMTC cells [92]

Imatinib (STI571) is a TKI that inhibits RET PDGFRand KIT A phase II trial in which imatinib was tested inmetastatic MTC patients yielded disappointing results Thepatients showed no objective responses however a minorityof patients achieved disease stability [93]

Several TK inhibitors have been tested in clinical trialsbut the most effective inhibitor and whether there is anyspecificity for particular RET mutations remain unknownA recent study compared the effects of four TKIs (axitinibsunitinib vandetanib and XL184) on cell proliferation RETexpression and autophosphorylation and ERK activation incell lines that express MEN2A (MTC-TT) andMEN2B (MZ-CRC-1) mutation The findings showed that the inhibitorswere specific for different mutations with XL184 being themost potent inhibitor against the MEN2A mutation andvandetanib the most effective against the MEN2B mutationin vitro No TK inhibitor was superior for all tested celllines which indicates that mutation-specific therapies couldbe beneficial in MTC treatment [94]

32 Other Emerging Therapies for Medullary Thyroid Cancer

321 Sensitization of Medullary Thyroid Carcinoma to Con-ventional Cytotoxic Treatments Conventional chemotherapyhas shown limited efficacy against metastatic MTC One ofthe mechanisms for the resistance of MTC to chemothera-peutic drugs is multidrug resistance (MDR) [95] MDR incancer cells has been attributed to the overexpression of


several plasma membrane ATP-dependent efflux pumpssuch as MDR-1 [96] The enzyme cyclooxygenase (COX-2)has been shown to regulate MDR-1 expression in rat mesan-gial cells [97] Furthermore a study has shown that in vitrotreatment of anMTC-derived cell line with rofecoxib a COX-2 inhibitor was able to sensitize MTC cells to doxorubicin[98] A recent study performed in vitro has shown thatcelecoxib another COX-2 inhibitor was able to induce bothMTC cell apoptosis and sensitization to vinorelbine thusenhancing the chemotherapeutic effect of this drug [99]A clinical trial in which the in vivo activities of celecoxibwere explored in MTC patients who cannot benefit fromavailable treatments would be desirable after accounting forthe possible cardiovascular risks of this drug

322 Drugs That Inhibit MTC Tumorigenesis Targets otherthan TKRs Several other therapeutic agents are being inves-tigated for their uses in the treatment of thyroid carcinomasincludingMTCThese agents inhibit targets that are involvedin development of MTC other than the tyrosine kinasereceptors We previously discussed that RAS operates ina complex signaling network with multiple activators andeffectors which allows them to regulate many cellular func-tions such as cell proliferation differentiation apoptosis andsenescence Phosphatidylinositol 3-kinase (PI3K) is one ofthe main effector pathways of RAS regulating cell growthcell cycle entry cell survival cytoskeleton reorganizationand metabolism The presence of Ras mutations in spo-radic MTC and most importantly the frequent activationof the PI3KAKTmTOR pathway in several aggressive andmetastatic MTCs strongly suggest that this cellular signalingpathway is a good candidate for targeted therapies againstMTC In fact several in vitro evidences have demonstratedthat the indirect blocking of this pathway by PI3K inhibitors[100] or direct inhibition of mTOR [48] can mediate theinduction of apoptosis and a decrease in cell viability in theMTC TT cell line Currently there are several clinical trialsin which patients with radioiodine-refractory differentiatedand medullary thyroid carcinomas were recruited to testthe efficacy of everolimus (RAD001) a novel inhibitor ofmTOR in combination with other drugs (NCT01625520and NCT01270321) Everolimus has demonstrated antitumorefficacy in various cancer types including MTC Recentlyresearchers have developed a liposomal form of everolimusand have demonstrated the anticancer efficacy of this for-mulation against TT cells [101] Studies have shown thatmetformin can decrease the proliferation of cancer cellsthrough 51015840-AMP-activated protein kinase-(AMPK-) depen-dent inhibition of mTOR Some researchers have reportedgrowth inhibitory effects of metformin against MTC celllines (TT and MZ-CRC-1) which were attributable to thedownregulation of bothmTOR6SK and pERK signalingTheexpression of the molecular targets of metformin in humanMTC cells suggests that this drug may be potentially usefulin the treatment of MTC [49]

PI3K-AKT-mTOR and RAF-MEK-ERK signaling havebeen shown to be important in the resistance of thyroidcancer cells to apoptosis and the promotion of tumor pro-gression In this context targeted anti-VEGFR therapy or

RAF inhibition may be ineffective if PI3K signaling remainsintact Therefore two promising drugs RAF265 a RAFinhibitor that is active against VEGFR2 and BEZ235 a PI3Kinhibitor were tested alone and in combination in preclinicalMTCmodels that represented the key genotypes observed inrefractory thyroid cancers The study findings showed that acombination treatment with agents that inhibited both RAFand PI3K pathways strongly inhibited growth both in vitroand in vivo In addition the investigators showed for the firsttime that RAF265 potently inhibits the constitutively active119877119864119879119862634119882 a form of the kinase that is observed in MEN2A

[102]Persistent RET activation a frequent event inMTC leads

to the activation of the PI3KAKTmTOR ERKMAPK andJAKSTAT3 pathways Recently the efficacy of the JAK12inhibitor AZD1480 against the growth of thyroid cancerwas tested in vitro and in vivo in thyroid cancer cell linesthat expressed oncogenic RET The findings showed thatAZD1480 efficiently inhibited the growth and tumorigenesisof thyroid cancer cell lines that harbored oncogenic RETalterations likely through the inhibition of PI3K-AKT signal-ing this result supports the use of this inhibitor in patientswith thyroid cancer particularly in those with advancedMTC for whom there are no effective therapeutic options[103]

Several MTCs display a rich but heterogeneous expres-sion of somatostatin receptors (sst1ndash5) [104] Thus somato-statin (SRIF) peptide analogues in combination with TKIsmay be a promising approach for the treatment of thesetumors The presence of different combinations of SRIFreceptor (SSTR) subtypes in a given patient may explain thevariable clinical response to SRIF analogues andmaypromotethe search for more selective drugs with different affinitiesto the various receptor subtypes [105] The currently avail-able somatostatin analogues (octreotide and lanreotide) actpreferentially through the somatostatin receptor subtype 2(sst2) In MTC these compounds have been reported toexert antisecretive effects on calcitonin but unfortunatelyare not reported to have antiproliferative effects Pasireotide(SOM230) is a new somatostatin analogue that has a peculiarbinding profile with high affinity for sst1 sst2 sst3 andsst5 Preliminary data from a phase II study of patients withmetastatic carcinoma show that SOM230 is effective andsome clinical trials are exploring the efficacy of SOM230alone or in combination with RAD001 in patients with MTC(NCT01625520 and NCT01270321)

The NF-120581B pathway is also a potential target for drugdevelopment and a number of compounds have been devel-oped to inhibit this pathway at different levels in cancer cellsStudies have demonstrated that the proteosome inhibitorbortezomib exerted a promising antitumor effect in humanMTC cells through the inhibition of I120581B degradation whichled to the inactivation of the transcriptional factor NF-120581B[106] Patients are currently being recruited for a phase IIItrial to study the combination of vandetanib plus bortezomib(httpwwwClinicalTrialsgov) PatientswithMTCwill par-ticipate in the phase II study [21]


323 Immunotherapy andRadioimmunotherapy Immuniza-tion with tumor antigen-pulsed autologous dendritic cells(DCs) resulted in protective immunity and the rejection ofvarious established human tumors Specifically vaccinationimmunotherapy with calcitonin andor carcinoembryonicantigen (CEA) peptide-pulsedDCswas shown to result in theinduction of a cellular antigen-specific immune response inpatients with MTC which led to clinical responses in somepatientsTherefore for the first time a potential DC vaccina-tion therapy was developed for patients with metastatic MTC[107] Another study which was performed in a transgenicMTC mouse model has confirmed this finding [108]

Papewalis et al have reported on the in vitro findings ofa vaccination trial in 5 MTC patients who were treated withDCs that were generated using a new protocol which con-sisted of granulocyte-macrophage colony-stimulating factorand interferon-alpha (IFN-DCs)These investigators demon-strated that immunization with IFN-DCs led to a tumorepitope-specificTh1 immune response inMTCpatients [109]Furthermore a pilot trial of 10 patients has assessed thesafety and efficacy in terms of immune responses and clinicalactivities of the DCs In this study DCs were injected intogroin lymph nodes at 3-week intervals Monitoring of thepatients included serial measurements of calcitonin tumormarkers radiological imaging and immunological in vitrotests including T-cell interferon-gamma detection and cyto-toxicity assays DC vaccinations were determined to be welltolerated and safe After a median followup of 11 months(range 7ndash26) 3 (30) of the 10 patients exhibited diseasestability while 7 (70) of the patients progressed duringtreatment A combined treatment with different tumor celllysate-pulsed DCs increased the likelihood of a calcitonintumor marker response and should therefore be preferredover monotherapy with DCs pulsed with a single lysate [110]

Thus immunotherapy may be a promising alternativetherapy in combination with agents that target specific signaltransduction pathways for aggressive forms of MTC that areresistant to classical therapies

A significant antitumor effect was also observed withradioimmunotherapy using an anti-CEA 131I-F6 monoclonalantibody in MTC-bearing nude mice Nevertheless nocomplete responses were observed Similarly to chemother-apy drugs that target the tumor microenvironment mightimprove the efficacy of radioimmunotherapyThis hypothesiswas confirmed by a recent study in which pretreatment ofmice grafted with the TT human medullary thyroid cancercell line with antiangiogenic therapies was found to improvethe efficacy of radioimmunotherapy with acceptable toxicity[111] Another independent study which was conducted ina mouse model of MTC has found similar results with theangiogenesis inhibitor bevacizumab [112] Future investiga-tions will be performed to better understand how antiangio-genic agents enhance the efficacy of radioimmunotherapy

324 Epigenetic Therapy Epigenetic drugs are expected totarget the two main mechanisms of epigenetic alterationsDNA methylation and acetylation and are regarded withincreasing interest by both endocrinologists and oncologists

Concluded trials of such drugs have shown that few patientswith advanced thyroid cancer responded completely whichsuggests that these treatments were effective at stabilizingprogressive disease Therefore definitive results from clinicaltrials will clarify the true effectiveness of epigenetic drugs inthese tumors Epigenetic drugs when used in combinationwith other target molecules might significantly increase res-ponse rates to treatment in advanced thyroid cancer patientseither by relaxing the chromatin structure to make DNAmore accessible to the effects of a DNA targeting drug orby acting synergistically with antimitotic drugs [113] Thusepigenetic therapymay be a promising novel approach for thetreatment of some cases of MTC

33 Potential Mechanisms of Resistance to Therapy in MTCTumor cells often devise strategies to bypass the effects ofantineoplastic agents and the selection of therapy-resistantclones is frequently the reason for treatment failure Onecharacteristic of endocrine cancer is a general resistance toconventional chemotherapies or radiotherapies that wouldnormally lead to apoptosis of the cancer cells MTC candevelop resistances to cytotoxic drugs due to the expressionof the multidrug resistance (MDR) 1 gene Drugs that canoppose this mechanism of resistance to traditional chemo-therapies may increase the sensitivity of MTC tumor cellsto chemotherapy For example celecoxib a COX-2 inhibitorhas been shown to potentiate the chemotherapeutic effect ofvinorelbine in MTC [99] Therefore the synergistic actionsof a cytotoxic drug and a compound that increases thesensitivity of MTC cells to such a drug could provide atreatment method for patients who cannot benefit fromTKIs However the most promising results in patients withchemotherapy and radiotherapy-unresponsive MTC wereobtained with TKIs The inhibition of a single RTK mayengage compensatory signaling that maintains cell growthMultitargeted tyrosine kinase inhibitors which inhibit mul-tiple RTKs simultaneously including RET have been devel-oped to bypass this potential resistance mechanism Onecomplication is that such inhibitors may not only be moreeffective for targeting receptors in tumor cells but may alsoexhibit greater toxicity Thus the challenge is to balance theincreased efficacy of these inhibitors with the potential fora broader array of side effects [7] Moreover some MTCpatients cannot take advantage of these therapies due tospecific RET mutations that confer resistance to TKIs (egRET V804 confers resistance to vandetanib) [76 114] Finallyan important study reported the existence of cancer stem-like cells in MTC which exhibit the features of self-renewaland of multiple lineage differentiation that is dependent onRET proto-oncogene receptor activity suggesting a potentialmechanism of resistance to cytotoxic or TKIs agents [115]

4 Conclusions

Most cases of metastatic MTC are incurable due to resis-tance to conventional chemo- and radiotherapies In recentdecades the identification of genetic defects and alteredcellular signaling pathways involved in humanMTC tumori-genesis have led to the development of targeted therapies


of which the most important are tyrosine kinase inhibitorsHowever the low rates of partial responses or complete res-ponses and the short duration of responses in MTC patientswho were observed while taking these drugs promptedresearchers to develop new drugs and alternative therapiesto be combined with multi-TKIs and to reduce potentialcross-toxicity effects To this end a recent study has shownsynergistic effects with a combination of sorafenib and theMEK inhibitor AZD6244 against a human MTC cell lineConcomitant use of a RAF inhibitor RAF265 and a dualPI3KmTOR inhibitor BEZ-235 was another effective com-binatorial therapy against thyroid cancer in xenograft mousemodels [102] The mTOR cascade is emerging probably asone of the most important deregulated pathways in advancedand metastatic MTC and certainly deserves further studyTargeting mTOR in combination might be efficacious inpatients with this tumor types

However a complete overview of all signaling pathwaysincluding their interactions role of the activating gene muta-tions that contribute toMTC tumorigenesis andmechanismsof intrinsic and acquired resistance to treatments is requiredand will permit us to identify the best therapy for eachpatient Novel combined or sequential therapies request afurther step in the knowledge of the cellular signaling ofthis tumor Finally larger multi-institutional clinical trials tofully understand the clinical benefits of these therapies arewarranted

Conflict of Interests

The authors do not have any conflict of interests to disclose

Acknowledgments

The authors are grateful to the Associazione Italiana Ricercasul Cancro theAmericanThyroidAssociation and the Foun-dations Sandro Pitigliani and Centro Oncologico Fiorentino

References

[1] A Antonelli SMartina Ferrari P Fallahi et al ldquoMedullary thy-roid cancer new targeted molecular therapiesrdquo Recent Patentson Endocrine Metabolic and Immune Drug Discovery vol 4 no1 pp 10ndash14 2010

[2] DW Ball S B Baylin and A C De Butros ldquoMedullary thyroidcarcinomardquo inWerner and Ingbarrsquos theThyroid L E Bravermanand R D Utiger Eds pp 930ndash943 Lippincott Williams andWilkins Philadelphia Pa USA 8th edition 2000

[3] A Pinchera ldquoMedullary thyroid cancer diagnosis and treat-mentrdquo in Practical Management ofThyroid Cancer l M ErnestC H Ujjal K Mallick and P Kendall-Taylor Eds pp 255ndash280Springer-Verlag Ltd London UK 2006

[4] S Roman R Lin and J A Sosa ldquoPrognosis of medullarythyroid carcinoma demographic clinical and pathologic pre-dictors of survival in 1252 casesrdquoCancer vol 107 no 9 pp 2134ndash2142 2006

[5] L Santarpia L Ye andR F Gagel ldquoBeyondRET potential ther-apeutic approaches for advanced and metastatic medullary

thyroid carcinomardquo Journal of Internal Medicine vol 266 no1 pp 99ndash113 2009

[6] M Brassard and G Rondeau ldquoRole of vandetanib in the man-agement of medullary thyroid cancerrdquo Biologics vol 6 pp 59ndash66 2012

[7] L Ye L Santarpia and R F Gagel ldquoThe evolving field of tyro-sine kinase inhibitors in the treatment of endocrine tumorsrdquoEndocrine Reviews vol 31 no 4 pp 578ndash599 2010

[8] M Husain R N Alsever and J P Lock ldquoFailure of medullarycarcinoma of the thyroid to respond to doxorubicin therapyrdquoHormone Research vol 9 no 1 pp 22ndash25 1978

[9] J P Droz M Schlumberger P Rougier M Ghosn P Gardetand C Parmentier ldquoChemotherapy in metastatic nonanaplasticthyroid cancer experience at the Institut Gustave-RoussyrdquoTumori vol 76 no 5 pp 480ndash483 1990

[10] H Scherubl F Raue and R Ziegler ldquoCombination chemother-apy of advanced medullary and differentiated thyroid cancerPhase II studyrdquo Journal of Cancer Research and Clinical Oncol-ogy vol 116 no 1 pp 21ndash23 1990

[11] A Antonelli P Fallahi S M Ferrari et al ldquoRET TKI potentialrole in thyroid cancersrdquo Current Oncology Reports vol 14 no2 pp 97ndash104 2012

[12] M B Lodish and C A Stratakis ldquoRET oncogene in MEN2MEN2B MTC and other forms of thyroid cancerrdquo ExpertReview of Anticancer Therapy vol 8 no 4 pp 625ndash632 2008

[13] M SAiraksinen andM Saarma ldquoTheGDNF family signallingbiological functions and therapeutic valuerdquo Nature ReviewsNeuroscience vol 3 no 5 pp 383ndash394 2002

[14] M Drosten and B M Putzer ldquoMechanisms of disease cancertargeting and the impact of oncogenic RET for medullarythyroid carcinoma therapyrdquo Nature Clinical Practice Oncologyvol 3 no 10 pp 564ndash574 2006

[15] Z Liu P Hou M Ji et al ldquoHighly prevalent genetic alter-ations in receptor tyrosine kinases and phosphatidylinositol 3-kinaseAkt and mitogen-activated protein kinase pathways inanaplastic and follicular thyroid cancersrdquo Journal of ClinicalEndocrinology and Metabolism vol 93 no 8 pp 3106ndash31162008

[16] C S Mitsiades V Kotoula V Poulaki et al ldquoEpidermal growthfactor receptor as a therapeutic target in human thyroid carci-noma mutational and functional analysisrdquo Journal of ClinicalEndocrinology and Metabolism vol 91 no 9 pp 3662ndash36662006

[17] BM Erovic D Kim C Cassol et al ldquoPrognostic and predictivemarkers in medullary thyroid carcinomardquo Endocrine Pathologyvol 23 no 4 pp 232ndash242 2012

[18] C Rodrıguez-Antona J Pallares C Montero-Conde et alldquoOverexpression and activation of EGFR and VEGFR2 inmedullary thyroid carcinomas is related to metastasisrdquo Endo-crine-Related Cancer vol 17 no 1 pp 7ndash16 2010

[19] M Croyle N Akeno J A Knauf et al ldquoRETPTC-induced cellgrowth is mediated in part by epidermal growth factor receptor(EGFR) activation evidence for molecular and functionalinteractions between RET and EGFRrdquo Cancer Research vol 68no 11 pp 4183ndash4191 2008

[20] C Capp S M Wajner D R Siqueira B A Brasil L Meurerand A L Maia ldquoIncreased expression of vascular endothelialgrowth factor and its receptors VEGFR-1 and VEGFR-2 inmedullary thyroid carcinomardquo Thyroid vol 20 no 8 pp 863ndash871 2010

[21] R S Kerbel ldquoTumor angiogenesisrdquo New England Journal ofMedicine vol 358 no 19 pp 2039ndash2049 2008


[22] K Gomez J Varghese and C Jimenez ldquoMedullary thyroid car-cinomamolecular signaling pathways and emerging therapiesrdquoJournal of Thyroid Research vol 2011 Article ID 815826 2011

[23] M Jeffers M Fiscella C P Webb M Anver S Koochekpourand G F Vande Woude ldquoThe mutationally activated Metreceptor mediates motility and metastasisrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 95 no 24 pp 14417ndash14422 1998

[24] M Papotti M OliveroM Volante et al ldquoExpression of hepato-cyte growth factor (HGF) and its receptor (MET) in medullarycarcinoma of the thyroidrdquo Endocrine Pathology vol 11 no 1 pp19ndash30 2000

[25] V M Wasenius S Hemmer M L Karjalainen-Lindsberg NN Nupponen K Franssila and H Joensuu ldquoMET receptortyrosine kinase sequence alterations in differentiated thyroidcarcinomardquo American Journal of Surgical Pathology vol 29 no4 pp 544ndash549 2005

[26] M Ivan J A Bond M Prat P M Comoglio and D Wynford-Thomas ldquoActivated ras and ret oncogenes induce over-expres-sion of c-met (hepatocyte growth factor receptor) in humanthyroid epithelial cellsrdquoOncogene vol 14 no 20 pp 2417ndash24231997

[27] R S Bernard L Zheng W Liu D Winer S L Asa and SEzzat ldquoFibroblast growth factor receptors as molecular targetsin thyroid carcinomardquo Endocrinology vol 146 no 3 pp 1145ndash1153 2005

[28] S Ezzat P Huang A Dackiw and S L Asa ldquoDual inhibitionof RET and FGFR4 restrains medullary thyroid cancer cellgrowthrdquo Clinical Cancer Research vol 11 no 3 pp 1336ndash13412005

[29] K Matsuo S H Tang B Sharifi S A Rubin R Schreck andJ A Fagin ldquoGrowth factor production by human thyroid car-cinoma cells abundant expression of a platelet-derived growthfactor-B-like protein by a human papillary carcinoma cell linerdquoJournal of Clinical Endocrinology and Metabolism vol 77 no 4pp 996ndash1004 1993

[30] A Cerrato V De Falco and M Santoro ldquoMolecular genetics ofmedullary thyroid carcinoma the quest for novel therapeutictargetsrdquo Journal of Molecular Endocrinology vol 43 no 4 pp143ndash155 2009

[31] D J Harrison M L Hooper J F Armstrong and A R ClarkeldquoEffects of heterozygosity for the Rb-1(t19neo) allele in themouserdquo Oncogene vol 10 no 8 pp 1615ndash1620 1995

[32] B O Williams L Remington D M Albert S Mukai RT Bronson and T Jacks ldquoCooperative tumorigenic effects ofgermline mutations in Rb and p53rdquo Nature Genetics vol 7 no4 pp 480ndash484 1994

[33] M Harvey H Vogel E Y H P Lee A Bradley and L ADonehower ldquoMice deficient in both p53 and Rb develop tumorsprimarily of endocrine originrdquo Cancer Research vol 55 no 5pp 1146ndash1151 1995

[34] A B Coxon J M Ward J Geradts G A Otterson M Zajac-Kaye and F J Kaye ldquoRET cooperates with RBp53 inactivationin a somatic multi-step model for murine thyroid cancerrdquoOncogene vol 17 no 12 pp 1625ndash1628 1998

[35] P P Joshi M V Kulkarni B K Yu et al ldquoSimultaneous down-regulation of CDK inhibitors p18Ink4c and p27Kip1 is requiredfor MEN2A-RET-mediated mitogenesisrdquoOncogene vol 26 no4 pp 554ndash570 2007

[36] C Segouffin-Cariou and M Billaud ldquoTransforming ability ofMEN2A-RET requires activation of the phosphatidylinositol 3-kinaseAKT signaling pathwayrdquo Journal of Biological Chemistryvol 275 no 5 pp 3568ndash3576 2000

[37] S C Pitt and H Chen ldquoThe phosphatidylinositol 3-kinaseaktsignaling pathway in medullary thyroid cancerrdquo Surgery vol144 no 5 pp 721ndash724 2008

[38] M A Kouvaraki C Liakou A Paraschi et al ldquoActivation ofmTOR signaling in medullary and aggressive papillary thyroidcarcinomasrdquo Surgery vol 150 no 6 pp 1258ndash1265 2011

[39] N Asai H Murakami T Iwashita and M Takahashi ldquoA muta-tion at tyrosine 1062 in MEN2A-Ret and MEN2B-Ret impairstheir transforming activity and association with Shc adaptorproteinsrdquo Journal of Biological Chemistry vol 271 no 30 pp17644ndash17649 1996

[40] H Murakami T Iwashita N Asai et al ldquoEnhanced phos-phatidylinositol 3-kinase activity and high phosphorylationstate of its downstream signalling molecules mediated by Retwith the MEN 2B mutationrdquo Biochemical and BiophysicalResearch Communications vol 262 no 1 pp 68ndash75 1999

[41] D Salvatore R M Melillo C Monaco et al ldquoIncreased in vivophosphorylation of ret tyrosine 1062 is a potential pathogeneticmechanism of multiple endocrine neoplasia type 2Brdquo CancerResearch vol 61 no 4 pp 1426ndash1431 2001

[42] G Pelicci F Troglio A Bodini et al ldquoThe neuron-specificRai (ShcC) adaptor protein inhibits apoptosis by coupling retto the phosphatidylinositol 3-kinaseAkt signaling pathwayrdquoMolecular and Cellular Biology vol 22 no 20 pp 7351ndash73632002

[43] I Sansal and W R Sellers ldquoThe biology and clinical relevanceof the PTEN tumor suppressor pathwayrdquo Journal of ClinicalOncology vol 22 no 14 pp 2954ndash2963 2004

[44] K M Zbuk and C Eng ldquoCancer phenomics RET and PTENas illustrative modelsrdquo Nature Reviews Cancer vol 7 no 1 pp35ndash45 2007

[45] T L Yuan and L C Cantley ldquoPI3K pathway alterations in can-cer variations on a themerdquo Oncogene vol 27 no 41 pp 5497ndash5510 2008

[46] I Rapa E Saggiorato D Giachino et al ldquoMammalian targetof rapamycin pathway activation is associated to RET mutationstatus in medullary thyroid carcinomardquo Journal of ClinicalEndocrinology and Metabolism vol 96 no 7 pp 2146ndash21532011

[47] A Tamburrino A A Molinolo P Salerno et al ldquoActivation ofthe mTOR pathway in primary medullary thyroid carcinomaand lymph node metastasesrdquo Clinical Cancer Research vol 18no 13 pp 3532ndash3540 2012

[48] S Grozinsky-Glasberg H Rubinfeld Y Nordenberg et alldquoThe rapamycin-derivative RAD001 (everolimus) inhibits cellviability and interacts with the Akt-mTOR-p70S6K pathwayin human medullary thyroid carcinoma cellsrdquo Molecular andCellular Endocrinology vol 315 no 1-2 pp 87ndash94 2010

[49] J Klubo-Gwiezdzinska K Jensen J Costello et al ldquoMetformininhibits growth and decreases resistance to anoikis inmedullarythyroid cancer cellsrdquo Endocrine Related Cancer vol 19 no 3 pp447ndash456 2012

[50] M Bockhorn A Frilling V Kalinin S Schroder and C EBroelsch ldquoAbsence of H- and K-ras oncogenemutations in spo-radic medullary thyroid carcinomardquo Experimental and ClinicalEndocrinology and Diabetes vol 108 no 1 pp 49ndash53 2000

[51] H J Schulten J Al-Maghrabi K Al-Ghamdi et al ldquoMuta-tional screening of RET HRAS KRAS NRAS BRAF AKT1


and CTNNB1 in medullary thyroid carcinomardquo AnticancerResearch vol 31 no 12 pp 4179ndash4183 2011

[52] RCiampi CMian L Fugazzola et al ldquoEvidence of a lowpreva-lence of Ras mutations in a large medullary thyroid cancerseriesrdquoThyroid vol 23 no 1 pp 50ndash57 2013

[53] M M Moura B M Cavaco A E Pinto and V LeiteldquoHigh prevalence of RAS mutations in RET-negative sporadicmedullary thyroid carcinomasrdquo Journal of Clinical Endocrinol-ogy and Metabolism vol 96 no 5 pp E863ndashE868 2011

[54] N Agrawal Y Jiao M Sausen et al ldquoExomic sequencingof medullary thyroid cancer reveals dominant and mutuallyexclusive oncogenic mutations in RET and RASrdquo Journal ofClinical Endocrinology and Metabolism 2012

[55] A Boichard L Croux A Al Ghuzlan et al ldquoSomatic Rasmutations occur in a large proportion of sporadic RET-negativemedullary thyroid carcinomas and extend to a previouslyunidentified exonrdquo The Journal of Clinical Endocrinology andMetabolism vol 97 no 10 pp E2031ndashE2035 2012

[56] R A Weinberg ldquoThe cat and mouse games that genes virusesand cells playrdquo Cell vol 88 no 5 pp 573ndash575 1997

[57] L Santarpia SM Lippman andAK El Naggar ldquoTargeting theMAPK-RAS-RAF signaling pathway in cancer therapyrdquo ExpertOpinion onTherapeutic Targets vol 16 no 1 pp 103ndash119 2012

[58] E B Carson M McMahon S B Baylin and B D NelkinldquoRet gene silencing is associated with raf-1-induced medullarythyroid carcinoma cell differentiationrdquoCancer Research vol 55no 10 pp 2048ndash2052 1995

[59] S ShirasawaM Furuse N Yokoyama and T Sasazuki ldquoAlteredgrowth of human colon cancer cell lines disrupted at activatedKi-rasrdquo Science vol 259 no 5104 pp 85ndash88 1993

[60] K W Wood H Qi G DrsquoArcangelo R C Armstrong TM Roberts and S Halegoua ldquoThe cytoplasmic raf oncogeneinduces a neuronal phenotype in PC12 cells a potential rolefor cellular raf kinases in neuronal growth factor signal trans-ductionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 90 no 11 pp 5016ndash5020 1993

[61] J I Park C J Strock D W Ball and B D Nelkin ldquoTheRasRafMEKextracellular signal-regulated kinase pathwayinduces autocrine-paracrine growth inhibition via the leukemiainhibitory factorJAKSTAT pathwayrdquo Molecular and CellularBiology vol 23 no 2 pp 543ndash554 2003

[62] D Arthan S K Hong and J I Park ldquoLeukemia inhibitory fac-tor can mediate RasRafMEKERK-induced growth inhibitorysignaling in medullary thyroid cancer cellsrdquo Cancer Letters vol297 no 1 pp 31ndash41 2010

[63] MKunnimalaiyaan AMVaccaroMANdiaye andH ChenldquoInactivation of glycogen synthase kinase-3120573 a downstreamtarget of the raf-1 pathway is associated with growth sup-pression in medullary thyroid cancer cellsrdquo Molecular CancerTherapeutics vol 6 no 3 pp 1151ndash1158 2007

[64] T S GujralW VanVeelen D S Richardson et al ldquoA novel RETkinase-120573-catenin signaling pathway contributes to tumorigen-esis in thyroid carcinomardquo Cancer Research vol 68 no 5 pp1338ndash1346 2008

[65] L Ludwig H Kessler M Wagner et al ldquoNuclear factor-120581B isconstitutively active in C-cell carcinoma and required for RET-induced transformationrdquo Cancer Research vol 61 no 11 pp4526ndash4535 2001

[66] L Santarpia and G Bottai ldquoInhibition of RET activated path-ways novel strategies for therapeutic intervention in humancancersrdquo Current Pharmaceutical Design vol 19 2012

[67] P M Lorusso and J P Eder ldquoTherapeutic potential of novelselective-spectrum kinase inhibitors in oncologyrdquo Expert Opin-ion on Investigational Drugs vol 17 no 7 pp 1013ndash1028 2008

[68] S Walsh R Prichard and A D Hill ldquoEmerging therapies forthyroid carcinomardquo Surgeon vol 10 no 1 pp 53ndash58 2012

[69] D Vitagliano V De Falco A Tamburrino et al ldquoThe tyrosinekinase inhibitor ZD6474 blocks proliferation of RET mutantmedullary thyroid carcinoma cellsrdquo Endocrine-Related Cancervol 18 no 1 pp 1ndash11 2011

[70] J M Stommel A C Kimmelman H Ying et al ldquoCoactivationof receptor tyrosine kinases affects the response of tumor cellsto targeted therapiesrdquo Science vol 318 no 5848 pp 287ndash2902007

[71] A Ocana E Amir B Seruga and A Pandiella ldquoDo we haveto change the way targeted drugs are developedrdquo Journal ofClinical Oncology vol 28 no 24 pp e420ndashe421 2010

[72] R van Amerongen and A Berns ldquoTargeted anticancer ther-apies mouse models help uncover the mechanisms of tumorescaperdquo Cancer Cell vol 13 no 1 pp 5ndash7 2008

[73] Z A Knight H Lin and K M Shokat ldquoTargeting the cancerkinome through polypharmacologyrdquo Nature Reviews Cancervol 10 no 2 pp 130ndash137 2010

[74] S I Sherman ldquoTargeted therapies for thyroid tumorsrdquoModernPathology vol 24 no 2 pp S44ndashS52 2011

[75] R S Herbst J V Heymach M S OrsquoReilly A Onn and AJ Ryan ldquoVandetanib (ZD6474) an orally available receptortyrosine kinase inhibitor that selectively targets pathways crit-ical for tumor growth and angiogenesisrdquo Expert Opinion onInvestigational Drugs vol 16 no 2 pp 239ndash249 2007

[76] F Carlomagno T Guida S Anaganti et al ldquoDisease associatedmutations at valine 804 in the RET receptor tyrosine kinaseconfer resistance to selective kinase inhibitorsrdquo Oncogene vol23 no 36 pp 6056ndash6063 2004

[77] A De Luca A Carotenuto A Rachiglio et al ldquoThe role ofthe EGFR signaling in tumor microenvironmentrdquo Journal ofCellular Physiology vol 214 no 3 pp 559ndash567 2008

[78] S A Wells Jr J E Gosnell R F Gagel et al ldquoVandetanib forthe treatment of patients with locally advanced or metastatichereditary medullary thyroid cancerrdquo Journal of Clinical Oncol-ogy vol 28 no 5 pp 767ndash772 2010

[79] BG Robinson L Paz-Ares AKrebs J Vasselli andRHaddadldquoVandetanib (100 mg) in patients with locally advanced ormetastatic hereditary medullary thyroid cancerrdquo Journal ofClinical Endocrinology andMetabolism vol 95 no 6 pp 2664ndash2671 2010

[80] H Commander G Whiteside and C Perry ldquoVandetanib firstglobal approvalrdquo Drugs vol 71 no 10 pp 1355ndash1365 2011

[81] S A Wells Jr B G Robinson R F Gagel et al ldquoVandetanib inpatients with locally advanced or metastatic medullary thyroidcancer a randomized double-blind phase III trialrdquo Journal ofClinical Oncology vol 30 no 2 pp 134ndash141 2012

[82] R Kurzrock E E Cohen S I Sherman et al ldquoLong-termresults in a cohort of medullary thyroid cancer (MTC) patients(pts) in a phase I study with XL-184 (BMS 907351) an oralinhibitor of MET VEGFR2 and RETrdquo Journal of ClinicalOncology vol 28 Abstract 5502 p 15S 2010

[83] R Kurzrock S I Sherman D W Ball et al ldquoActivity of XL184(cabozantinib) an oral tyrosine kinase inhibitor in patientswith medullary thyroid cancerrdquo Journal of Clinical Oncologyvol 29 no 19 pp 2660ndash2666 2011


[84] F Carlomagno S Anaganti T Guida et al ldquoBAY 43-9006 inhi-bition of oncogenic RET mutantsrdquo Journal of the National Can-cer Institute vol 98 no 5 pp 326ndash334 2006

[85] E T Lam M D Ringel R T Kloos et al ldquoPhase II clinical trialof sorafenib in metastatic medullary thyroid cancerrdquo Journal ofClinical Oncology vol 28 no 14 pp 2323ndash2330 2010

[86] K Frank-Raue M Ganten M C Kreissl and F Raue ldquoRapidresponse to sorafenib in metastatic medullary thyroid carci-nomardquo Experimental and Clinical Endocrinology and Diabetesvol 119 no 3 pp 151ndash155 2011

[87] J Capdevila L Iglesias I Halperin et al ldquoSorafenib in patients(pts) with advanced thryroid carcinoma (TC) a compassionateuse programrdquo Journal of Clinical Oncology vol 28 Abstract5590 p 15S 2010

[88] L L Carr D A Mankoff B H Goulart et al ldquoPhase II studyof daily sunitinib in FDG-PET-positive iodine-refractory dif-ferentiated thyroid cancer and metastatic medullary carcinomaof the thyroid with functional imaging correlationrdquo ClinicalCancer Research vol 16 no 21 pp 5260ndash5268 2010

[89] A Ravaud C De La Fouchardiere J Asselineau et al ldquoEfficacyof sunitinib in advanced medullary thyroid carcinoma inter-mediate results of phase II THYSUrdquo Oncologist vol 15 no 2pp 212ndash213 2010

[90] A Polverino A Coxon C Starnes et al ldquoAMG 706 an oralmultikinase inhibitor that selectively targets vascular endothe-lial growth factor platelet-derived growth factor and kit recep-tors potently inhibits angiogenesis and induces regression intumor xenograftsrdquo Cancer Research vol 66 no 17 pp 8715ndash8721 2006

[91] M J Schlumberger R Elisei L Bastholt et al ldquoPhase II studyof safety and efficacy of motesanib in patients with progressiveor symptomatic advanced or metastatic medullary thyroidcancerrdquo Journal of Clinical Oncology vol 27 no 23 pp 3794ndash3801 2009

[92] A Coxon J Bready S Kaufman et al ldquoAnti-tumor activity ofmotesanib in a medullary thyroid cancer modelrdquo Journal ofEndocrinological Investigation vol 35 no 2 pp 181ndash190 2012

[93] JW B de Groot B A Zonnenberg P Q VanUfford-Mannesseet al ldquoA phase II trial of imatinib therapy for metastaticmedullary thyroid carcinomardquo Journal of Clinical Endocrinologyand Metabolism vol 92 no 9 pp 3466ndash3469 2007

[94] H H G Verbeek M M Alves J W B De Groot et al ldquoTheeffects of four different tyrosine kinase inhibitors on medul-lary and papillary thyroid cancer cellsrdquo Journal of Clinical Endo-crinology and Metabolism vol 96 no 6 pp E991ndashE995 2011

[95] C Massart J Gibassier C Lucas P Pourquier and J RobertldquoDoxorubicine resistancemodulation by ciclosporin a and vera-pamil in five human cell lines ormedullary thyroid cancerrdquo Bul-letin du Cancer vol 83 no 1 pp 39ndash45 1996

[96] A Breier M Barancık Z Sulova and B Uhrık ldquoP-glycopro-tein-Implications of metabolism of neoplastic cells and cancertherapyrdquoCurrent Cancer Drug Targets vol 5 no 6 pp 457ndash4682005

[97] V A Patel M J Dunn and A Sorokin ldquoRegulation of MDR-1 (P-glycoprotein) by cyclooxygenase-2rdquo Journal of BiologicalChemistry vol 277 no 41 pp 38915ndash38920 2002

[98] M C Zatelli A Luchin D Piccin et al ldquoCyclooxygenase-2 inhibitors reverse chemoresistance phenotype in medullarythyroid carcinoma by a permeability glycoprotein-mediatedmechanismrdquo Journal of Clinical Endocrinology and Metabolismvol 90 no 10 pp 5754ndash5760 2005

[99] A Vivaldi R Ciampi A Tacito et al ldquoColecoxib a cyclo-oxygenase-2 inhibitor potentiates the chemotherapic effect ofvinorelbine in the medullary thyroid cancer TT cell linerdquoMole-cular and Cellular Endocrinology vol 355 no 1 pp 41ndash48 2012

[100] M Kunnimalaiyaan M Ndiaye and H Chen ldquoApoptosis-mediated medullary thyroid cancer growth suppression by thePI3K inhibitor LY294002rdquo Surgery vol 140 no 6 pp 1009ndash1015 2006

[101] Y Iwase and Y Maitani ldquoPreparation and in vivo evaluationof liposomal everolimus for lung carcinoma and thyroid carci-nomardquo Biological amp Pharmaucetical Bulletin vol 35 no 6 pp975ndash979 2012

[102] N Jin T Jiang D M Rosen B D Nelkin and D W BallldquoSynergistic action of a RAF inhibitor and a dual PI3KmTORinhibitor in thyroid cancerrdquoClinical Cancer Research vol 17 no20 pp 6482ndash6489 2011

[103] J P Couto A Almeida L Daly M Sobrinho-Simoes J FBromberg and P Soares ldquoAZD1480 blocks growth and tumori-genesis of RET-activated thryoid cancer cell linesrdquo PloS Onevol 7 no 10 p E46869 2012

[104] M Papotti U Kumar M Volante C Pecchioni and Y CPatel ldquoImmunohistochemical detection of somatostatin recep-tor types 1ndash5 in medullary carcinoma of the thyroidrdquo ClinicalEndocrinology vol 54 no 5 pp 641ndash649 2001

[105] E Mato X Matıas-Guiu A Chico et al ldquoSomatostatin andsomatostatin receptor subtype gene expression in medullarythyroid carcinomardquo Journal of Clinical Endocrinology andMeta-bolism vol 83 no 7 pp 2417ndash2420 1998

[106] C S Mitsiades D McMillin V Kotoula et al ldquoAntitumoreffects of the proteasome inhibitor bortezomib in medullaryand anaplastic thyroid carcinoma cells in vitrordquo Journal ofClinical Endocrinology andMetabolism vol 91 no 10 pp 4013ndash4021 2006

[107] M Schott J Seissler M Lettmann V Fouxon W AScherbaum and J Feldkamp ldquoImmunotherapy for medullarythyroid carcinoma by dendritic cell vaccinationrdquo Journal ofClinical Endocrinology andMetabolism vol 86 no 10 pp 4965ndash4969 2001

[108] M Wuttke C Papewalis Y Meyer et al ldquoAmino acid-modified calcitonin immunization induces tumor epitope-specific immunity in a transgenic mouse model for medullarythyroid carcinomardquo Endocrinology vol 149 no 11 pp 5627ndash5634 2008

[109] C Papewalis M Wuttke B Jacobs et al ldquoDendritic cell vac-cination induces tumor epitope-specific Th1 immune responsein medullary thyroid carcinomardquo Hormone and MetabolicResearch vol 40 no 2 pp 108ndash116 2008

[110] T Bachleitner-Hofmann J Friedl M Hassler et al ldquoPilot trialof autologous dendritic cells loaded with tumor lysate(s) fromallogeneic tumor cell lines in patients withmetastaticmedullarythyroid carcinomardquo Oncology Reports vol 21 no 6 pp 1585ndash1592 2009

[111] F Kraeber-Bodere C Bodet-Milin C Niaudet et al ldquoCom-parative toxicity and efficacy of combined radioimmunother-apy and antiangiogenic therapy in carcinoembryonic antigen-expressing medullary thyroid cancer xenograftrdquo Journal ofNuclear Medicine vol 51 no 4 pp 624ndash631 2010

[112] P Y Salaun C Bodet-Milin E Frampas et al ldquoToxicity andefficacy of combined radioimmunotherapy and bevacizumab ina mouse model of medullary thyroid carcinomardquo Cancer vol116 no 4 pp 1053ndash1058 2010


[113] M G Catalano N Fortunati and G Boccuzzi ldquoEpigeneticsmodifications and therapeutic prospects in human thyroidcancerrdquo Frontiers in Endocrinology vol 3 p 40 2012

[114] C Romei S Mariotti L Fugazzola et al ldquoMultiple endocrineneoplasia type 2 syndromes (MEN 2) results from the ItaMENnetwork analysis on the prevalence of different genotypes andphenotypesrdquo European Journal of Endocrinology vol 163 no 2pp 301ndash308 2010

[115] W Zhu T Hai L Ye and G J Cote ldquoMedullary thyroid carci-noma cell lines contain a self-renewingCD133 + population thatis dependent on Ret proto-oncogene activityrdquo Journal of ClinicalEndocrinology andMetabolism vol 95 no 1 pp 439ndash444 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


to TKIs that target the RET and other cell receptors andthe activities of other signal transduction pathways that areinvolved in MTC tumorigenesis and progression but notdirectly targeted by TKIs In recent years the discovery ofmechanisms of resistance to TKIs and of several other mole-cular events that contribute to MTC transformation andmetastasis suggested that combinatory therapy may result ina more significant tumor growth inhibition This has led tothe development of novel compounds that have been used inseveral clinical trials including TKIs that can target multipleTKRs simultaneously in addition to RET and agents that cantarget other altered signaling pathways Other studies havedemonstrated the potential for immunotherapy in combi-nation with agents that target signal transduction pathwaysthat are important for MTC growth [5] Because the aim ofthese targeted therapies is to extend lifespan and increasethe quality of life it is very important to limit the toxicitiesof therapeutic agents either alone or in combination Thepossibility of testing these novel drugs in vitro (in primarythyroid cancer cells) and in vivo may help to improve thepersonalization of treatments [11]

2 Key Cellular Signaling Pathways andAlterations in MTC

21 RET Pathway The role of the RET oncogene in thetumorigenesis of MTC has been characterized extensively[12] The RET gene encodes a transmembrane tyrosinekinase that binds to glial cell line-derived neurotrophicfactor (GDNF) family ligands [13] RET signaling leads tothe activation of the RASmitogen-activated protein kinase(MAPK) and the phosphatidylinositol 31015840 kinase (PI3K)Aktpathways and has key roles in cell growth differentiationand survival Activating point mutations of the TKR REThave been reported in nearly all hereditary cases of MTCsome of these mutations are included in theMEN2A familialMTC or MEN2B syndromes in which there is a geno-typicphenotypic correlation between the type of RET muta-tion and clinical features RET mutations are also found in30ndash50 of sporadicMTCs Germlinemutations in the RETproto-oncogene are responsible for hereditary MTC whilesomatic RET mutations are responsible for sporadic MTC[14] These data provide a strong rationale for targeting RETin selective cancer therapy However this paper will mainlyfocus on additional cellular signaling pathways other thanRET responsible of MTC tumorigenesis and progression andpotential targeted approaches for the treatment of advancedor metastatic MTC

22 Additional Signaling PathwaysThat AccelerateMTC Prog-ression Although activating mutations of the TKR RET arebelieved to be the primary oncogenic event in the deve-lopment of amajority ofMTC cases it is clear that RET coop-erates with other signal transduction pathways to promoteMTC tumorigenesis

221 Tyrosine Kinase Receptors other than RETAre Implicatedin MTC Tumorigenesis In addition to RET other kinase

receptors may play a role in the development and progressionof MTCs [15]

Similar to the RET receptor the epidermal growth factorreceptor (EGFR) is a TKR that is associated with the regula-tion of cell growth proliferation and apoptosis Dimerizationof the receptor following ligand binding results in transphos-phorylation and the subsequent activation of several down-stream signal pathways EGFR has been shown to be fre-quently overexpressed in various types of thyroid carcinomasincluding MTC and to play a role in cancer developmentand progression [16] In contrast a recent report analyzingdifferent MTC on tissue microarrays has demonstrated thatonly 20 of cases revealed moderate to strong reactivity forEGFR whereas the majority of the cases revealed weak andvery focal positivity [17] A different study has shown thatEGFR-activating mutations are rare in MTC With respect tothe numbers of EGFR gene copies in MTCs the researchersdid not detect amplifications but did find polysomes in 15 ofthe examined tumors [18] Additionally EGFR was activatedin a subset of MTCs which suggests that this subset ofpatients might benefit from drugs that target also EGFRRecent findings have shown that the ligand-induced activa-tion of EGFR can stimulate RET activation beyond signalingand growth stimulation [19] Several EGFR inhibitors havebeen shown to markedly inhibit the growth of the MTC TTand MZ-CRC-1 cell lines Because RET activation seems tobe influenced by EGFR a recent study investigated whetherEGFR activation could be related to specific RET mutationsin MTCs The researchers found that tumors with the mostaggressive RET mutations (in codons 883918) exhibitedreduced EGFR expression compared to otherRETmutationsIt could be speculated that themost aggressiveRETmutationsare less dependent on EGFR activation [18] In fact in thework by Croyle et al [19] in which cell lines with RETmutations in codon 634 and codon 918 were compared theeffect of EGFR inhibition on the codon 918 mutated cell lineappeared to be reduced in agreement with the previous dataBecause the activation status of EGFR seems to be relatedto RET activation EGFR activation has been examined inRET-negative tumors [18] However no differences havebeen found in EGFR activation between RET-positive and-negative tumors which likely indicates that other molecularmechanisms lead to RET activation such as increased RETgene copy numbers altered promoter activity or increasedtranscription in the RET mutation-negative tumors Thesedata suggest that EGFR status determination in MTCs mightbe important but certainly deserves further investigations

The vascular endothelial growth factor receptor (VEGFR)pathway is also important in the pathogenesis of MTCThereare three transmembrane receptors that mediate the angio-genic and lymphogenic effects ofVEGFVEGFR-1 VEGFR-2and VEGFR-3 VEGFR-2 is thought to be implicated pri-marily in tumor growth and metastasis Overexpression ofVEGF and VEGFR-2 has been found in MTC compared tonormal thyroid tissue [20] The VEGF proteins (VEGF-A BC and D) which are secreted by tumor cells act as ligandsfor theVEGFR-2 receptors on endothelial cells and promote asignaling cascade through different pathways such as PLC-120574-PKC-Raf-MEK-MAPK and PI3K-Akt that stimulate cellular

































MA

PK p

athw

ay

MA

PK p

athw





Vandetanib


Everolimus

SorafenibCRAF


Proliferation

Differentiation


RETEGFR

MET

FGRFKIT

Thyroid tumor

cell

PTEN

Bortezomib


cell


cell

VEGFR3 VEGFR2PDGFR




Differentiation

IKK

PI3K

mTOR

MEK

RAS



ERK

BRAF

MEK

ERK

PI3K

AKT AKT

mTOR

BRAF

RASJAK

STAT

NF120581B

Growth tumor


Metastasis







Table1Maintargeted

therapiesc

urrentlyin

usefor

thetreatmento

fmedullary

thyroidcancer

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Vand

etanib

(ZD6474)

radicradic

radicradic

01

Bortezom

ib

Notyetrecruitin

gNCT

0166117

9(12)

Recruitin

gNCT

005140

46(12)

NCT

01496313

(4)


results

NCT

00410761

(3)

NCT

00358956

(2)

NCT

00098345

(2)

Activenotrecruiting

NCT

000923247(12)

NCT

01298323

(3)

With

draw

nDocetaxel

NCT

000937417

XL184

(Cabozantin

ib)radic

radicradic

radicradic

000

4

Activenotrecruiting

NCT

00704730

(3)

NCT

00215605

(1)

Available

NCT

01683110

MainTK

IsSorafenib

(BAY

-43-9006)radic

radicradic

radicradic

radicradic

00059ndash0

05

Activenotrecruiting

NCT

00390325

(2)

Unknow

nlowastlowast

NCT

006542387(2)

Sunitin

ib(SU11248)

radicradic

radicradic

radic022ndash13

Activenotrecruiting

NCT

00381641

(2)

NCT

00519896

(2)

NCT

005106

40(2)

Motesanib

(AMG-706)

radicradic

radicradic

005ndash0

09

Completed

NCT

00121628

(2)

Imatinib

(STI571)

radicradic

radic5ndash37

XelodaD

acarbazine

Activenotrecruiting

NCT

00354523

(12)


Table1Con

tinued

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Everolim

us(RAD001)

mTO

RPasireotid

ePasireotid

e

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

NCT

01164176

(2)

Unknow

nlowastlowast

NCT

01118

65(2)

Main

Non

-TKIs

Bortezom

ib(PS-341)

proteosome

Vand

etanib

Activenotrecruiting

NCT

00923247

(12)

Pasireotid

e(SOM230)

sst1

35(som

atostatin

receptors)

Everolim

usEv

erolim

us

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

lowastIC

50RE

T[120583M]50

inhibitory

concentration

lowastlowastStatus

hasn

otbeen

verifi

edin

morethantwoyears





























4 Conclusions







Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































MA

PK p

athw

ay

MA

PK p

athw





Vandetanib


Everolimus

SorafenibCRAF


Proliferation

Differentiation


RETEGFR

MET

FGRFKIT

Thyroid tumor

cell

PTEN

Bortezomib


cell


cell

VEGFR3 VEGFR2PDGFR




Differentiation

IKK

PI3K

mTOR

MEK

RAS



ERK

BRAF

MEK

ERK

PI3K

AKT AKT

mTOR

BRAF

RASJAK

STAT

NF120581B

Growth tumor


Metastasis







Table1Maintargeted

therapiesc

urrentlyin

usefor

thetreatmento

fmedullary

thyroidcancer

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Vand

etanib

(ZD6474)

radicradic

radicradic

01

Bortezom

ib

Notyetrecruitin

gNCT

0166117

9(12)

Recruitin

gNCT

005140

46(12)

NCT

01496313

(4)


results

NCT

00410761

(3)

NCT

00358956

(2)

NCT

00098345

(2)

Activenotrecruiting

NCT

000923247(12)

NCT

01298323

(3)

With

draw

nDocetaxel

NCT

000937417

XL184

(Cabozantin

ib)radic

radicradic

radicradic

000

4

Activenotrecruiting

NCT

00704730

(3)

NCT

00215605

(1)

Available

NCT

01683110

MainTK

IsSorafenib

(BAY

-43-9006)radic

radicradic

radicradic

radicradic

00059ndash0

05

Activenotrecruiting

NCT

00390325

(2)

Unknow

nlowastlowast

NCT

006542387(2)

Sunitin

ib(SU11248)

radicradic

radicradic

radic022ndash13

Activenotrecruiting

NCT

00381641

(2)

NCT

00519896

(2)

NCT

005106

40(2)

Motesanib

(AMG-706)

radicradic

radicradic

005ndash0

09

Completed

NCT

00121628

(2)

Imatinib

(STI571)

radicradic

radic5ndash37

XelodaD

acarbazine

Activenotrecruiting

NCT

00354523

(12)


Table1Con

tinued

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Everolim

us(RAD001)

mTO

RPasireotid

ePasireotid

e

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

NCT

01164176

(2)

Unknow

nlowastlowast

NCT

01118

65(2)

Main

Non

-TKIs

Bortezom

ib(PS-341)

proteosome

Vand

etanib

Activenotrecruiting

NCT

00923247

(12)

Pasireotid

e(SOM230)

sst1

35(som

atostatin

receptors)

Everolim

usEv

erolim

us

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

lowastIC

50RE

T[120583M]50

inhibitory

concentration

lowastlowastStatus

hasn

otbeen

verifi

edin

morethantwoyears





























4 Conclusions







Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Maintargeted

therapiesc

urrentlyin

usefor

thetreatmento

fmedullary

thyroidcancer

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Vand

etanib

(ZD6474)

radicradic

radicradic

01

Bortezom

ib

Notyetrecruitin

gNCT

0166117

9(12)

Recruitin

gNCT

005140

46(12)

NCT

01496313

(4)


results

NCT

00410761

(3)

NCT

00358956

(2)

NCT

00098345

(2)

Activenotrecruiting

NCT

000923247(12)

NCT

01298323

(3)

With

draw

nDocetaxel

NCT

000937417

XL184

(Cabozantin

ib)radic

radicradic

radicradic

000

4

Activenotrecruiting

NCT

00704730

(3)

NCT

00215605

(1)

Available

NCT

01683110

MainTK

IsSorafenib

(BAY

-43-9006)radic

radicradic

radicradic

radicradic

00059ndash0

05

Activenotrecruiting

NCT

00390325

(2)

Unknow

nlowastlowast

NCT

006542387(2)

Sunitin

ib(SU11248)

radicradic

radicradic

radic022ndash13

Activenotrecruiting

NCT

00381641

(2)

NCT

00519896

(2)

NCT

005106

40(2)

Motesanib

(AMG-706)

radicradic

radicradic

005ndash0

09

Completed

NCT

00121628

(2)

Imatinib

(STI571)

radicradic

radic5ndash37

XelodaD

acarbazine

Activenotrecruiting

NCT

00354523

(12)


Table1Con

tinued

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Everolim

us(RAD001)

mTO

RPasireotid

ePasireotid

e

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

NCT

01164176

(2)

Unknow

nlowastlowast

NCT

01118

65(2)

Main

Non

-TKIs

Bortezom

ib(PS-341)

proteosome

Vand

etanib

Activenotrecruiting

NCT

00923247

(12)

Pasireotid

e(SOM230)

sst1

35(som

atostatin

receptors)

Everolim

usEv

erolim

us

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

lowastIC

50RE

T[120583M]50

inhibitory

concentration

lowastlowastStatus

hasn

otbeen

verifi

edin

morethantwoyears





























4 Conclusions







Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Con

tinued

Keytargets


Drugname

IC50RE

T[120583M]lowast

RET

EGFR

VEG

FRs

PDGFR

cKIT

FGFR

MET

BRAF

Other

Drugs

incombinatio

nStatus

identifi

er(phase)

Everolim

us(RAD001)

mTO

RPasireotid

ePasireotid

e

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

NCT

01164176

(2)

Unknow

nlowastlowast

NCT

01118

65(2)

Main

Non

-TKIs

Bortezom

ib(PS-341)

proteosome

Vand

etanib

Activenotrecruiting

NCT

00923247

(12)

Pasireotid

e(SOM230)

sst1

35(som

atostatin

receptors)

Everolim

usEv

erolim

us

Recruitin

gNCT

01625520

(2)

NCT

01270321

(2)

lowastIC

50RE

T[120583M]50

inhibitory

concentration

lowastlowastStatus

hasn

otbeen

verifi

edin

morethantwoyears





























4 Conclusions







Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































4 Conclusions







Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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