breast & colon cancer: molecular alterations & therapeutic ... · the non-steroidal,...

Breast & Colon Cancer: Molecular Breast & Colon Cancer: Molecular Alterations & Therapeutic TargetsAlterations & Therapeutic TargetsPart 1Part 1

June 1, 2007June 1, 2007Nancy HynesNancy HynesFMIFMI

In the past 25 yrs cancer research has generated a largeIn the past 25 yrs cancer research has generated a largebody of knowledge on molecular mechanisms controlling allbody of knowledge on molecular mechanisms controlling allaspects of normal cellular physiology/signaling pathways. aspects of normal cellular physiology/signaling pathways.

These studies have also provided knowledge on molecules &These studies have also provided knowledge on molecules &mechanisms underlying cancer development. mechanisms underlying cancer development.

GoalGoal –– to design rational/molecularly targeted therapeutics to design rational/molecularly targeted therapeutics for cancer treatment.for cancer treatment.

Molecular targets for cancer therapyMolecular targets for cancer therapy

Major goal of lecturesMajor goal of lectures

Present studies from the area of breast & colon cancerPresent studies from the area of breast & colon cancerto show how molecular analyses can be used to achieve the to show how molecular analyses can be used to achieve the ultimate goal of providing better therapies for patients. ultimate goal of providing better therapies for patients.

Molecular targets for cancer therapyMolecular targets for cancer therapy

Breast cancerBreast cancer1 in 8 women develop the cancer1 in 8 women develop the cancer30% will die of 30% will die of metastaticmetastatic diseasedisease

--incidence has been decreasingincidence has been decreasing

--therapeutics targeting proteins involvedtherapeutics targeting proteins involvedin breast cancer are in clinical use in breast cancer are in clinical use

Colon cancerColon cancer3rd most common cancer3rd most common cancer

--therapeutics targeting proteins involved in colon cancer are intherapeutics targeting proteins involved in colon cancer are inpreclinical testing; drugs targeting angiogenesis have been apprpreclinical testing; drugs targeting angiogenesis have been approvedoved

P. Ravdin et al 2007 NEJM 356:1670

The original description of pathological stages of The original description of pathological stages of colorectal cancer as defined by Duke in 1932colorectal cancer as defined by Duke in 1932

Davies et al 2005 Nature Rev Cancer 5:199 Davies et al 2005 Nature Rev Cancer 5:199

Confined to bowel wall Confined to bowel wall –– no LN metastases no LN metastases

Lymph node metastasesLymph node metastases

Distant metastasesDistant metastases

The colorectal The colorectal adenomoadenomo--carcinoma sequencecarcinoma sequence

Davies et al 2005 Nature Rev Cancer 5:199 Davies et al 2005 Nature Rev Cancer 5:199

A well defined A well defined molecular/pathologicalmolecular/pathological sequence of colorectal cancersequence of colorectal cancerdevelopment has emerged over the past 15 yrs based on the development has emerged over the past 15 yrs based on the pioneering work of Vogelstein & colleagues pioneering work of Vogelstein & colleagues

Gene Expression Patterns & Breast Cancer Gene Expression Patterns & Breast Cancer

Gene expression profiling is a broad method for Gene expression profiling is a broad method for molecularly defining tumor types. molecularly defining tumor types.

Tumor subTumor sub--types with distinct gene expression patternstypes with distinct gene expression patternshave been described.have been described.

prognosisprognosisdiagnosisdiagnosisnew therapeutic targetsnew therapeutic targets

A well defined molecular/pathological sequence of A well defined molecular/pathological sequence of breast cancer is not available. breast cancer is not available.

Gene expression profiling & breast cancerGene expression profiling & breast cancer

Breast cancers fall into 5 clinicallyBreast cancers fall into 5 clinically--relevant subtypes*relevant subtypes*

T. Sorlie et al PNAS 2001 98:10869; ibid 2003 100:8413

Basal-like ErbB2+ Normal Luminal Type B Luminal Type A

*cDNA microarrays on a core set of 8100 genes were carried out, then 427 unique genes formed a basis for classification based upon significantly greater variation in expression between different tumors than between paired samples from same tumor.


Breast cancers fall into 5 clinicallyBreast cancers fall into 5 clinically--relevant subtypesrelevant subtypes

Disease-free survivalOverall survival

Luminal A

Basal-like

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++Luminal have best prognosisLuminal have best prognosisBasal have worst prognosisBasal have worst prognosis

What influences Breast Cancer development?What influences Breast Cancer development?

Genetic & Epigenetic AlterationsGenetic & Epigenetic Alterations

HormonesHormones

What influences breast cancer development?What influences breast cancer development?

Many of these factors are related to estrogen exposureMany of these factors are related to estrogen exposure

Estrogen & Breast CancerEstrogen & Breast Cancer

Estrogens play an essential role in development Estrogens play an essential role in development of the normal breast & in cancer.of the normal breast & in cancer.

The life time exposure to estrogens has a strong The life time exposure to estrogens has a strong influence on cancer development.influence on cancer development.

Early onset of menarche & late menopauseEarly onset of menarche & late menopause

Loss of ovarian function prevents breast cancerLoss of ovarian function prevents breast cancer

Low incidence of male breast cancerLow incidence of male breast cancer

Early fullEarly full--term pregnancy reduces risk of breastterm pregnancy reduces risk of breastcancer cancer (post(post--menopausal ER+ type).menopausal ER+ type).

Estrogen & Breast CancerEstrogen & Breast Cancer--TimelineTimeline

18961896George George BeatsonBeatson described that removal of the ovaries described that removal of the ovaries from women with from women with metastaticmetastatic breast cancer sometimesbreast cancer sometimesled to tumor regression. led to tumor regression.

““On the treatment of inoperable cases of carcinoma of theOn the treatment of inoperable cases of carcinoma of themamma: suggestions for a new method of treatment with mamma: suggestions for a new method of treatment with illustrative casesillustrative cases””.. Lancet (1896)Lancet (1896)

19621962Jensen & Jacobsen identified the target for estrogen action,Jensen & Jacobsen identified the target for estrogen action,the estrogen receptor (ER).the estrogen receptor (ER).

““Basic guides to the mechanism of estrogen actionBasic guides to the mechanism of estrogen action””Recent Recent ProgProg HormHorm Res (1962)Res (1962)

19711971Jensen correlated the presence of ER with hormone Jensen correlated the presence of ER with hormone responsiveness of the breast tumor. responsiveness of the breast tumor.

““Estrogen receptors & breast cancer response to Estrogen receptors & breast cancer response to adrenalectomyadrenalectomy””NatlNatl Cancer Inst Cancer Inst MonogrMonogr, 1971, 1971

The estrogen receptor as target for therapyThe estrogen receptor as target for therapy

ERER++ tumors depend upon estrogen for proliferation.tumors depend upon estrogen for proliferation.

ER antagonists were some of the first targeted, rationalER antagonists were some of the first targeted, rationaltherapeutics .therapeutics .(proposed in 1936 by (proposed in 1936 by LacassagneLacassagne))

In 1971 Jensen correlated the presence of ER with hormone In 1971 Jensen correlated the presence of ER with hormone responsiveness of the breast tumor. responsiveness of the breast tumor.

Many primary breast tumors are ERMany primary breast tumors are ER++

and depend upon ER action for growth.and depend upon ER action for growth.

Taken from Smith and Dowsett, 2003

The estrogen receptor as a therapeutic target The estrogen receptor as a therapeutic target in breast cancerin breast cancer

The nonThe non--steroidal, antisteroidal, anti--estrogenestrogenTamoxifenTamoxifen has been used since has been used since 1973 for breast cancer therapy. 1973 for breast cancer therapy.

TamoxifenTamoxifen blocks ER blocks ER transcriptional activity transcriptional activity

Unfortunately after a few years most breast cancer patientsUnfortunately after a few years most breast cancer patientsrelapse & show metastases during treatment with relapse & show metastases during treatment with tamoxifentamoxifen..

Why?Why?Has proliferation become independent of ER action? Has proliferation become independent of ER action?

In postmenopausal women, adrenal & ovarian androgens In postmenopausal women, adrenal & ovarian androgens are converted into estrogens, in different peripheral are converted into estrogens, in different peripheral tissues: muscle, skin, normal breast & in breast cancer tissues: muscle, skin, normal breast & in breast cancer cells, by the enzyme cells, by the enzyme aromatasearomatase. .

AromataseAromatase inhibitors & breast cancer inhibitors & breast cancer

Taken from Smith and Dowsett, 2003

The nonThe non--steroidal, antisteroidal, anti--estrogenestrogenTamoxifenTamoxifen has been used since has been used since 1973 for breast cancer therapy. 1973 for breast cancer therapy.

TamoxifenTamoxifen blocks ER blocks ER transcriptional activity transcriptional activity

LetrazoleLetrazole

AIsAIs lower lower estradiolestradiolproduction. production.

AromataseAromatase inhibitors inhibitors were recently approvedwere recently approvedfor clinical use.for clinical use.

The estrogen receptor as a therapeutic target The estrogen receptor as a therapeutic target in breast cancerin breast cancer

Aromatase inhibitors lower the level of estrogens in the skin, adiposetissue & the tumor; this lowers ER activity & blocks cancer cell proliferation.

AromataseAromatase inhibitors & breast cancer inhibitors & breast cancer

Some have shown efficacy in patients who relapsed on Some have shown efficacy in patients who relapsed on tamoxifentamoxifen therapy. therapy.

These results suggest that tumors still require These results suggest that tumors still require estrogen/ER for proliferation; lowering the level of estrogen/ER for proliferation; lowering the level of estrogen in the tumor & adipose tissue has an impact on estrogen in the tumor & adipose tissue has an impact on disease.disease.

These results are very important for breast cancer These results are very important for breast cancer patients:patients:

1 1 –– after relapse on after relapse on tamoxifentamoxifen there is another there is another therapeutic option with proven efficacy therapeutic option with proven efficacy

2 2 –– antianti--estogenestogen therapy is relatively nontherapy is relatively non--toxic toxic

Why does resistance emerge?Why does resistance emerge?More laterMore later……

Ultimately most ER+ breast cancer patients that respondUltimately most ER+ breast cancer patients that respondto antito anti--estrogen based therapies (estrogen based therapies (tamoxifentamoxifen or AI)or AI)relapse on treatment. relapse on treatment.

ClinincalClinincal problems problems ––antihormonalantihormonal therapiestherapies

What influences breast cancer development?What influences breast cancer development?

Genetic & Epigenetic AlterationsGenetic & Epigenetic Alterations

HormonesHormones

Specific Molecular Alterations & Therapies Specific Molecular Alterations & Therapies

Development of targeted therapeutics based uponDevelopment of targeted therapeutics based uponcharacterizing specific molecular alterations in tumors.characterizing specific molecular alterations in tumors.e.g., ErbB2 gene amplificatione.g., ErbB2 gene amplification

loss of tumor suppressor genesloss of tumor suppressor genesDNA or transcriptional level DNA or transcriptional level

activation of dominant activation of dominant oncogenesoncogenes::mutation, amplification, transcriptionalmutation, amplification, transcriptional

Tumor suppressor genes & breast cancerTumor suppressor genes & breast cancer

BRCA 1 & BRCA2BRCA 1 & BRCA2

Existence of genes responsible for inherited predispositionExistence of genes responsible for inherited predispositionto breast & ovarian cancer was suggested more than to breast & ovarian cancer was suggested more than 100 yrs ago.100 yrs ago.

Due to foresight of certain human geneticists, Due to foresight of certain human geneticists, G G LeniorLenior &&MC KingMC King, who collected material from , who collected material from ““cancer familiescancer families””, , linkage analyses were used to determine chromosomallinkage analyses were used to determine chromosomallocation of the genes, this was followed by their location of the genes, this was followed by their positional cloning. positional cloning.

What characterizes a family with What characterizes a family with hereditary breast cancer?hereditary breast cancer?

1010--15% of all breast & ovarian cancers are inherited15% of all breast & ovarian cancers are inheritedBRCA1 & BRCA2 are responsible for <50% of these. BRCA1 & BRCA2 are responsible for <50% of these.

The BRCA proteins have been implicated in many The BRCA proteins have been implicated in many different processes including different processes including DNA repairDNA repair & & recombination, cell cycle control & transcription. recombination, cell cycle control & transcription.

What are the functions of BRCA1 & BRCA2? What are the functions of BRCA1 & BRCA2?

The BRCA proteins are important for The BRCA proteins are important for the maintenance of genomic stability the maintenance of genomic stability

Large proteins with many proteinLarge proteins with many protein--protein interaction domainsprotein interaction domains--

Mouse cells deficient in BRCA2 show spontaneous Mouse cells deficient in BRCA2 show spontaneous aberrations in chromosome structure.aberrations in chromosome structure.

metaphase spreadmetaphase spread

Aberrations in the usual Aberrations in the usual UU--shaped mouse chromosomes shaped mouse chromosomes are visible.are visible.(enlarged on the right)(enlarged on the right)

chromatidchromatid breakbreak

triradialtriradial &&quatraradialquatraradialstructuresstructures

VenkitaramanVenkitaraman 2002 Cell 108: 1712002 Cell 108: 171

BRCA proteins & DNA RepairBRCA proteins & DNA Repair

A cell undergoes 10A cell undergoes 10--20 double strand breaks/day 20 double strand breaks/day --in addition to other types of DNA damage.in addition to other types of DNA damage.

single strand annealingsingle strand annealing homologous recombinationhomologous recombination


In S phase In S phase -- HR is the preferred repair pathwayHR is the preferred repair pathway


nonhomologousnonhomologous end joiningend joining


nonhomologousnonhomologous end joiningend joiningsingle strand annealingsingle strand annealing homologous recombinationhomologous recombination

The preferred errorThe preferred error--freefreeHR pathway does not HR pathway does not fxnfxn in cellsin cellslacking BRCA proteins.lacking BRCA proteins.

A cell undergoes 10A cell undergoes 10--20 double strand breaks/day 20 double strand breaks/day --in addition to other types of DNA damage.in addition to other types of DNA damage.


BRCA1 has multiple functionsBRCA1 has multiple functions

BRCA1 has a broad role linked to a range of processesBRCA1 has a broad role linked to a range of processes--implicated in different cell cycle checkpoints.implicated in different cell cycle checkpoints.

A. Ashworth August 2005 Cancer Cell A. Ashworth August 2005 Cancer Cell

SS--phase: phase: Repair of DS DNA breaks by Repair of DS DNA breaks by homologous recombinationhomologous recombination

G2/M:G2/M:DNA DNA decatenationdecatenation to separate to separate entangled chromosomesentangled chromosomes

BRCA1 has multiple functionsBRCA1 has multiple functions

BRCA1 has a broad role linked to a range of processesBRCA1 has a broad role linked to a range of processes--implicated in different cell cycle checkpoints.implicated in different cell cycle checkpoints.

A. Ashworth August 2005 Cancer Cell A. Ashworth August 2005 Cancer Cell

SS--phase: phase: Repair of DS DNA breaks by Repair of DS DNA breaks by homologous recombinationhomologous recombination

G2/M:G2/M:DNA DNA decatenationdecatenation to separate to separate entangled chromosomesentangled chromosomes

BRCA2 has a more specific role BRCA2 has a more specific role in repair of DNA DS breaks. in repair of DNA DS breaks.

1 1

22

33

44

ATM & ATR ATM & ATR kinaseskinases are activatedare activatedin response to DNA DS breaks. in response to DNA DS breaks.

ATM & ATR P multiple proteins ATM & ATR P multiple proteins -- tototransducetransduce the DS break response signal. the DS break response signal.

Cell cycle is stalledCell cycle is stalled--via CH2 & CHK1via CH2 & CHK1

DNA repair stimulatedDNA repair stimulatedNHEJ or HRNHEJ or HR

Cellular response to DS DNA breaksCellular response to DS DNA breaks-- 4 steps 4 steps

BRCA2, Rad51 & DNA repair BRCA2, Rad51 & DNA repair

BRCA2 is required for the regulation of RAD51BRCA2 is required for the regulation of RAD51--mediated homologous mediated homologous recombinationalrecombinational repair.repair.

F. F. EsashiEsashi et al 2007 Nature et al 2007 Nature StructStruct & Mol & Mol BiolBiol 21 May 2007 doi:10.1038/nsmb124521 May 2007 doi:10.1038/nsmb1245

Rad51Rad51 Rad51Rad51 Rad51Rad51Rad51Rad51 Rad51Rad51Rad51Rad51

Rad51Rad51Rad51Rad51


Rad51 monomers bind the BRC motifsRad51 monomers bind the BRC motifs

Rad51 filaments bind the CRad51 filaments bind the C--Terminal Domain (CTD) Terminal Domain (CTD)

Ser3291Ser3291--PP

blocks Rad51 blocks Rad51 associationassociation


F. F. EsashiEsashi et al 2007 Nature et al 2007 Nature StructStruct & Mol & Mol BiolBiol 21 May 2007 doi:10.1038/nsmb124521 May 2007 doi:10.1038/nsmb1245

Rad51Rad51 Rad51Rad51 Rad51Rad51Rad51Rad51 Rad51Rad51Rad51Rad51


Rad51Rad51Rad51Rad51Rad51 monomers bind the BRC motifsRad51 monomers bind the BRC motifs

Rad51 filaments bind the CRad51 filaments bind the C--Terminal domain (CTD)Terminal domain (CTD)

Ser3291Ser3291--PP

Rad51 Rad51 filament filament associationassociation

Site is deSite is de--P in P in response to IRresponse to IR


CTD association of Rad51 filaments facilitates efficient CTD association of Rad51 filaments facilitates efficient nucleactionnucleaction of RAD51 of RAD51 multimersmultimers on DNA on DNA –– HR is stimulated HR is stimulated

Based on the knowledge of the roles of BRCA proteins in DNA repaBased on the knowledge of the roles of BRCA proteins in DNA repair ir the next goal was to design inhibitors that would specifically kthe next goal was to design inhibitors that would specifically kill ill tumor cells with BRCA mutations tumor cells with BRCA mutations -- and spare normal cellsand spare normal cells. .

SyntheticSynthetic lethalitylethality::A A genegene ((pathwaypathway) ) thatthat has no has no phenotypephenotype whenwhen deleteddeleted ((inhibitedinhibited) ) alonealone isis mademade essential essential byby lossloss of a of a redundant redundant partnerpartner ((pathwaypathway).).

Targeting BRCA tumors Targeting BRCA tumors

R.KennedyR.Kennedy & & A.DA.D’’AndreaAndrea 2006 JCO 24:37992006 JCO 24:3799

Targeting compensatory pathways in BRCA Targeting compensatory pathways in BRCA deficient tumors deficient tumors

N.TurnerN.Turner et al 2005 et al 2005 CurrCurr OpinOpin in Pharmacology 5:388in Pharmacology 5:388

SpecificSpecific DNA DNA lesionslesions oror lesionslesionsthatthat block block oror cause cause thethe collapsecollapseof of replicationreplication forksforks areare veryverydependentdependent on on HRHR--basedbased DNA DNA repairrepair..

SSBsSSBs areare repairedrepaired byby basebase excisionexcisionrepairrepair pathwaypathway; ; inhibitioninhibition of of thisthispathwaypathway increasesincreases thethe numbernumber of of unrepairedunrepaired SSBsSSBs leadingleading to to collapsecollapse of of replicationreplication forksforks & & increaseincrease in DSBs at in DSBs at replicationreplicationforksforks..

poly(ADPpoly(ADP--riboseribose) ) polymerasepolymerase(PARP1) (PARP1) isis criticalcritical forfor basebaseexcisionexcision repairrepair..

Nature 2005 Nature 2005 volvol 434, p913 & p917434, p913 & p917

Targeting BRCA tumors Targeting BRCA tumors

Based on the knowledge of the roles of BRCA proteins in DNA repaBased on the knowledge of the roles of BRCA proteins in DNA repair ir the next goal was to design inhibitors that would specifically kthe next goal was to design inhibitors that would specifically kill ill tumor cells with BRCA mutations tumor cells with BRCA mutations -- and spare normal cellsand spare normal cells. .

KU005864 PARP1 inhibitor KU005864 PARP1 inhibitor –– IC50 = 3.2 IC50 = 3.2 nMnM

H.FarmerH.Farmer et al 2005 Nature 434: 917.et al 2005 Nature 434: 917.

Targeting BRCA tumors with a PARP inhibitor Targeting BRCA tumors with a PARP inhibitor

PARP inhibition selectively blocks the in vivo growth of PARP inhibition selectively blocks the in vivo growth of BRCA2BRCA2--deficient tumors & does not affect growth of WT tumors.deficient tumors & does not affect growth of WT tumors.

In the absence of BRCA1 or BRCA2, PARP inhibition leads to In the absence of BRCA1 or BRCA2, PARP inhibition leads to persistent SS gaps in DNA that collapse into DS breaks at persistent SS gaps in DNA that collapse into DS breaks at a replication fork. Repair of these breaks by errora replication fork. Repair of these breaks by error--prone prone DSB repair mechanisms causes many aberrations that DSB repair mechanisms causes many aberrations that eventually lead to cell death. eventually lead to cell death.

BRCABRCA--2 KO tumors + KU00586842 KO tumors + KU0058684

BRCABRCA--2 KO tumors + Vehicle control2 KO tumors + Vehicle control

WT cells + KU0058684WT cells + KU0058684

WT cells + Vehicle controlWT cells + Vehicle control

Various PARP inhibitors are in clinical development Various PARP inhibitors are in clinical development

AstraZenecaAstraZeneca


BRCA1 tumors have a basal cancer BRCA1 tumors have a basal cancer ““expression signatureexpression signature””

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++

Would PARP inhibition also inhibit growth of tumors with a BasalWould PARP inhibition also inhibit growth of tumors with a Basalphenotype? Are there sporadic cancers with BRCA1 mutations phenotype? Are there sporadic cancers with BRCA1 mutations –– in in the basalthe basal--like group?like group?

(Turner et al 2004 Nature Reviews Cancer vol 4)

BRCA1 & BRCA2 have not been implicated in BRCA1 & BRCA2 have not been implicated in sporadic breast cancersporadic breast cancer

In contrast to other inherited tumor suppressor genes In contrast to other inherited tumor suppressor genes (APC in colon cancer), sporadic breast cancers do not (APC in colon cancer), sporadic breast cancers do not carry mutations in BRCA genes. carry mutations in BRCA genes.

Other mechanisms contribute to loss of BRCA expression Other mechanisms contribute to loss of BRCA expression or function in breast cancer. or function in breast cancer.

BRCA1 transcription is silenced by promoter BRCA1 transcription is silenced by promoter methylationmethylation in sporadic breast & ovarian cancers. in sporadic breast & ovarian cancers.

Epigenetic mechanisms of BRCA1 inactivationEpigenetic mechanisms of BRCA1 inactivation

Turner et al 2004 Nature Reviews Cancer vol 4

Mutations in BRCA2 interacting proteinsMutations in BRCA2 interacting proteins

T.Walsh & MC.King 2007 Cancer Cell 11:103

PALB2 binds N-terminus of BRCA2& its binding is essential for BRCA2 DSB DNA repair activity.

PALB2 mutations in familial & sporadic breast cancersPALB2 mutations in familial & sporadic breast cancers

FrameshiftFrameshift mutation found in 113 families with history mutation found in 113 families with history of breast & ovarian cancer.of breast & ovarian cancer.

Same mutation found in 18/1918 sporadic breast cancers.Same mutation found in 18/1918 sporadic breast cancers.

Important for two reasons:Important for two reasons:~50% of inherited breast cancer is due to unknown mutations.~50% of inherited breast cancer is due to unknown mutations.

Evidence that the BRCA pathway/complex is involved in Evidence that the BRCA pathway/complex is involved in sporadic breast/ovarian cancer. sporadic breast/ovarian cancer.

PALB2 mutations in familial & sporadic breast cancersPALB2 mutations in familial & sporadic breast cancers

p165p165

Identified PALB truncating mutationsin 10/923 individuals with familialbreast cancer (0/1084 controls).

Mutations in BRCA2 interacting proteinsMutations in BRCA2 interacting proteins

T.Walsh & MC.King 2007 Cancer Cell 11:103

Proteins in red carry germlinemutations that predispose tobreast cancer.

Proteins aberrantly expressed or activatedProteins aberrantly expressed or activatedin breast cancerin breast cancer

ErbB2 & EGF receptor tyrosine ErbB2 & EGF receptor tyrosine kinaseskinases

cc--mycmyc

Cyclin D1Cyclin D1

FGF receptor tyrosine FGF receptor tyrosine kinaseskinases

…………

Hynes et al 1989Hynes et al 1989

ErbB2 ErbB2 overexpressionoverexpression in primary breast cancer in primary breast cancer

ERBB2ERBB2 gene amplification was the first consistent gene amplification was the first consistent genetic alteration found in breast cancer (20genetic alteration found in breast cancer (20--25%).25%).

Patients with high ErbB2 tend to have a poor clinical outcome. Patients with high ErbB2 tend to have a poor clinical outcome.


Disease-free survivalOverall survival

Luminal A

ErbB2+Basal-like

BasalBasal NormalNormal Luminal BLuminal B Luminal ALuminal AErbB2ErbB2++

ErbB2+ group has a bad prognosisErbB2+ group has a bad prognosis

PPPPP

PPPPP

PPPPP

PPPPP

MAPK

AKT

STAT

SRC

mTOR

PPPPPPPPPP

PPPPP

PPPPP

PPPPP

PPPPPPPPPP

Cytoplasmic DomainCytoplasmic DomainPPPPP

PPPPPPPPPPPPPPP

PPPPP PPPPPPI3K

ExtracellularExtracellular DomainDomain

Plasma MembranePlasma Membrane

ERBB Receptors

X

Z

ERBB targeted antibodies:HerceptinCetuximab

TKI

TKI

ERBB targeted tyrosine kinaseinhibitors (TKIs):GefitinibErlotinib

ERBB ERBB targetedtargeted inhibitorsinhibitors

Herceptin was approved for treatment of Herceptin was approved for treatment of metastaticmetastaticErbB2 ErbB2 overexpressingoverexpressing breast cancer patients in 1998.breast cancer patients in 1998.

Slamon et al NEJM 2001 vol 344

Median time to disease progression

7.4 mos.

4.6 mos.

15 patients-CR6 patients-CR

HerceptinHerceptin®®//TrastuzumabTrastuzumab & Breast Cancer& Breast Cancer

Herceptin®® does not cure metastatic breast cancer –at this stage tumor cells have spread to distant organs.

HerceptinHerceptin®® & Breast Cancer& Breast Cancer

Once HerceptinOnce Herceptin®® was approved for treatment of advanced was approved for treatment of advanced metastaticmetastatic breast cancer patients, it was tested on+breast cancer patients, it was tested on““earlyearly--stagestage”” ErbB2ErbB2+ breast cancer patients, in combinationbreast cancer patients, in combinationwith chemotherapy.with chemotherapy.

Patients all had ErbB2 Patients all had ErbB2 overexpressionoverexpression & most were lymph node& most were lymph node++, , i.ei.e, the tumor had spread to , the tumor had spread to LNsLNs and possibly to distant organs. and possibly to distant organs.

Results from trials testing Results from trials testing HerceptinHerceptin® in the adjuvant setting* in the adjuvant setting* showed that its addition to chemotherapy reduced the risk of showed that its addition to chemotherapy reduced the risk of recurrence by 50% in women with early stage ErbB2recurrence by 50% in women with early stage ErbB2++ cancer.cancer.((RomondRomond et al 2005 N et al 2005 N EnglEngl J Med 353:1673; PiccardJ Med 353:1673; Piccard--GebhartGebhart et al ibid pg 1659)et al ibid pg 1659)

*adjuvant setting *adjuvant setting -- directly after surgical removal of the tumordirectly after surgical removal of the tumor

2005 NEJM vol 353

Addition of trastuzumabreduced mortalityrate by one third.

These results suggest that trastuzumab acts as a chemosensitizer when given together with chemotherapy directly after surgery.

In vitro ErbB2In vitro ErbB2++ model to model to test DNA repair in the absence test DNA repair in the absence or presence of or presence of trastuzumabtrastuzumab. .

TrastuzumabTrastuzumab blocks the ability blocks the ability of ErbB2of ErbB2++ tumor cells to repairtumor cells to repairciscis--platinplatin induced DNA damage. induced DNA damage.

Why is this important? Why is this important?

Blocking ErbB2 with HerceptinBlocking ErbB2 with Herceptin®® ((TKIsTKIs ?) may make the ?) may make the tumor cells very sensitive to chemotherapeutics.tumor cells very sensitive to chemotherapeutics.

ErbB2ErbB2++ tumors & DNA repair tumors & DNA repair

ErbB2ErbB2+ + Cancer CellCancer Cell

Elevated ErbB2Elevated ErbB2++

SignalingSignalingPathway 1Pathway 1

Akt, ERK, Akt, ERK, mTORmTORetc etc


?? ??

Survival Survival

ErbB2ErbB2++ tumors & DNA repair tumors & DNA repair

ErbB2ErbB2+ + Cancer CellCancer Cell

Elevated ErbB2Elevated ErbB2++


Akt, ERK, Akt, ERK, mTORmTORetc etc


?? ??

Survival Survival

HerceptinHerceptin

ChemotherapeutiChemotherapeutidrugsdrugs

ChemotherapeuticChemotherapeuticdrugsdrugs

??

??

Drugs tested with HerceptinDrugs tested with Herceptin®

In clinical trialsIn clinical trials:: In vitro:In vitro:Doxorubicin (Topo2 inhibitorDoxorubicin (Topo2 inhibitor--blocksblocks CisCis--platinplatin (DNA cross(DNA cross--linker,linker,

replication by intercalation)replication by intercalation) alkylatingalkylating agent)agent)CyclophosphamideCyclophosphamide ((alkylatingalkylating agent)agent)PaclitaxelPaclitaxel (MT stabilizer)(MT stabilizer)

ERBB Receptors and Breast Cancer:ERBB Receptors and Breast Cancer:Clinical Problems to SolveClinical Problems to Solve

Understand mechanisms underlyingUnderstand mechanisms underlyingactivity of ERBB targeted inhibitors.activity of ERBB targeted inhibitors.

What mechanisms contribute What mechanisms contribute to resistance? to resistance? June 21June 21stst--Cellular SignalingCellular Signaling

ERBB inhibitors in combination with ERBB inhibitors in combination with other antiother anti--cancer drugs. Best choice?cancer drugs. Best choice?

PPPPP

PPPPP

PPPPP

PPPPP

MAPK

AKT

STAT

SRCmTOR

PPPPPPPPPP PPPPP

PPPPP

PPPPP

PPPPPPPPPP

PPPPPPPPPP

PPPPPPPPPP

PPPPP PPPPPPI3KX

ZTKI

TKI

ERBB targeted antibodies:HerceptinCetuximab

TKIs:GefitinibErlotinib

ERBB receptors & resistance to antiERBB receptors & resistance to anti--ERERtargeted therapies.targeted therapies.

--ERBB2 expression levels increase in tumors at time ofERBB2 expression levels increase in tumors at time ofrelapse on tamoxifen. relapse on tamoxifen.

(Dowsett & colleagues (Dowsett & colleagues -- Gutierrez et al 2005 JCO 23:2469)Gutierrez et al 2005 JCO 23:2469)

Mechanisms contributing to Mechanisms contributing to TamoxifenTamoxifen resistanceresistance

-- ERBB2 gene amplification observed in some patients who were ErbERBB2 gene amplification observed in some patients who were ErbB2B2--

at the start of at the start of TamoxifenTamoxifen treatment. treatment. Dowsett & colleagues Dowsett & colleagues -- Gutierrez et al 2005 JCO 23:2469Gutierrez et al 2005 JCO 23:2469


FISH FISH + = ErbB2 gene amplification= ErbB2 gene amplification

Estrogen receptor has multiple P sites.Estrogen receptor has multiple P sites.

ER ER –– P sitesP sites

Ser 104 Cdk2Ser 104 Cdk2Ser 118 ERK 1/ 2, cdk7 & othersSer 118 ERK 1/ 2, cdk7 & othersSer 167 p90 RSK & AKTSer 167 p90 RSK & AKTSer 115 MEKK1 (H Lee et al 2000 Ser 115 MEKK1 (H Lee et al 2000 MolMol Endo 14)Endo 14)ThrThr 311 p38 MAPK (H Lee et al 2002 MCB 22)311 p38 MAPK (H Lee et al 2002 MCB 22)Ser 305 PKA (R Ser 305 PKA (R MichalidesMichalides et al 2004 Cancer Cell)et al 2004 Cancer Cell)


Estrogen receptor has multiple P sites.Estrogen receptor has multiple P sites.

When signaling pathway activity is elevatedWhen signaling pathway activity is elevated-- e.g., in ErbB2 e.g., in ErbB2 overexpressingoverexpressing tumors cells, these sites are P.tumors cells, these sites are P.

ER transcriptional activity is highER transcriptional activity is high––even in the absence of estrogens. even in the absence of estrogens.

Elevated activity of other Elevated activity of other RTKsRTKs: IGFR: IGFR--1 or FGFR also impact1 or FGFR also impacton ER activity & are likely to play a role in the developmenton ER activity & are likely to play a role in the developmentof of tamoxifentamoxifen resistance. resistance.

P.Carter P.Carter volvol 1, p1181, p118

AntiAnti--Tumor Approaches using Antibodies Tumor Approaches using Antibodies

W. Wels et al 1992 W. Wels et al 1992 Cancer Res 52:6310Cancer Res 52:6310--17.17.

scFvscFv--toxintoxin

Structure of Single Chain Antibody Structure of Single Chain Antibody -- scFvscFv

Fv

W.Wels

HybridomaHybridoma cells producing cells producing FRP5FRP5, a , a murinemurine mAbmAb that binds thethat binds theextracellularextracellular domain of human ErbB2, were used to isolate domain of human ErbB2, were used to isolate cDNAscDNAsencoding the specific heavy and light chain variable domains.encoding the specific heavy and light chain variable domains.I.I.--M. M. HarwerthHarwerth et al. 1992 JBC 267:15160et al. 1992 JBC 267:15160--67. 67.

TargetingTargeting ErbB2 ErbB2 withwith specificspecific scFvscFv toxinstoxins

(W. Wels et al 1992 Cancer Res)

Exotoxin A from Pseudomonas aeruginosa

ChimericChimeric scFvscFv--Toxin Fusion ProteinToxin Fusion Protein

W.Wels

scFvscFv--Toxin Binds ErbB2 & Internalizes into Tumor CellsToxin Binds ErbB2 & Internalizes into Tumor Cells

ToxinToxin’’s Receptors Receptor ErbB2ErbB2

W.Wels

Internalized ErbB2 Internalized ErbB2 enters the enters the endosomesendosomes

scFvscFv--Toxin Specifically Kills ErbB2Toxin Specifically Kills ErbB2++ CellsCells

W.Wels

ErbB2ErbB2--Overexpressing Tumor XenograftsOverexpressing Tumor Xenograftsare Sensitive to FRP5are Sensitive to FRP5--ETA ETA

W.Wels

Recombinant scFv(FRP5)Recombinant scFv(FRP5)--ETA was produced as an experimental ETA was produced as an experimental drug under GMP conditions & provided by Ciba drug under GMP conditions & provided by Ciba GeigyGeigy AG, Basel. AG, Basel.

(Azemar et al 2003 Breast Cancer Res Treatment)

Compassionate Use Clinical Trial Compassionate Use Clinical Trial

G2M Cancer Drugs AG, Frankfurt

n=114 CR2 PR

Phase l dosePhase l dose--finding study finding study

Results:N = 172 Stable disease3 Clinical activity11 Disease progression

Goals: - determination of MTD - dose-limiting toxicity of IV injected scFv(FRP5)-ETA

Protocol: - 5 daily infusions – 2 consecutive weeks- 5 doses

von Minckwitz et al 2005 Breast Cancer Res 7:R617

Licensed to TopoTarget A/S Denmark in 2005Novartis licensed scFv(FRP5)-ETA to G2M Cancer Drugs AG, Frankfurt

Phase l dosePhase l dose--finding study finding study

Major conclusions:

- scFv-ETA can be safely administered IV at doses up to 12.5 μg/ml per day

- potentially therapeutic serum concentrations were reached

- major dose-limiting side effect was hepatotoxicity

von Minckwitz et al 2005 Breast Cancer Res 7:R617

breast & colon cancer: molecular alterations & therapeutic ... · the non-steroidal,...

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