metastasis and angiogenesis.2009 - amazon s3€¦ · 4/6/09 6 genes involved in metastatic steps,...

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MetastasisandAngiogenesis

AdvancedTopicsinCancerBiology2009

Sears

Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

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•  Benign tumors generally do not spread by invasion or metastasis

•  Malignant tumors are capable of spreading by invasion and metastasis – leading cause of cancer deaths

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Invasion and Metastasis •  Abnormal cells proliferate

and spread (metastasize) to other parts of the body

•  Invasion - direct migration and penetration into neighboring tissues

•  Metastasis - cancer cells penetrate into lymphatic system and blood vessels

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Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

A Role for EMT in The Metastatic Process

PolyakandWeinberg,2009

#Theepithelial–mesenchymaltransiEon(EMT)istriggeredbyadiversesetofsEmuliincludinggrowthfactorsignalling,tumour–stromalcellinteracEonsandhypoxia.#EMThasbeenshowntoresultincancercellswithstemcell‐likecharacterisEcsthathaveapropensitytoinvadesurroundingEssueanddisplayresistancetocertaintherapeuEcintervenEons.#Themesenchymal–epithelialtransiEon(MET)mayhavearoleinthereversionofdisseminatedmesenchymaltumourcellstoamoreepithelialstateindistantmetastases.#microRNAshavebeenidenEfiedasanewclassofEMTregulators,inpartowingtotheirregulaEonofEMT‐inducingtranscripEonfactors.

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Signaling networks regulating EMT

PolyakandWeinberg,2009

*

KeyTxnFactors

PolyakandWeinberg,2009

Expression and clinical relevance of selected EMT-associated genes

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Snail-associated epithelial-mesenchymal transition promotes oesophageal squamous cell carcinoma motility and progression

Y Usami 1 2, S Satake 1, F Nakayama 1, M Matsumoto 1, K Ohnuma 1, T Komori 2, S Semba 1, A Ito 1, H Yokozaki 1 *

epithelial‐typeOESCCcells

2008J.Pathology

TheEpithelial‐MesenchymalTransiEonGeneratesCellswithProperEesofStemCells

SenduraiA.Mani1,3,9,GoToCorrespondingAuthor,,WenjunGuo1,9,Mai‐JingLiao1,9,ElinorNg.Eaton1,AyyakkannuAyyanan4,AliciaY.Zhou1,2,MaryBrooks1,FerencReinhard1,ChengChengZhang1,MichailShipitsin5,6,LaurenL.Campbell5,7,KorneliaPolyak5,6,7,Cathrin

Brisken4,JingYang8andRobertA.Weinberg1,2

CellsInjected TumorsIncidence/NumberofInjecEons 1X106 1X105 1X104 1X103 HMLE‐Vector‐Ras 2/6 3/9 0/9 0/9 HMLE‐Snail‐Ras 6/6 9/9 9/9 6/9 HMLE‐Twist‐Ras 6/6 9/9 9/9 7/9

Table2TumorIncidenceofTransformedHMLEsInducedtoUndergoEMTbyEctopicExpressionofSnailorTwistandThenInjectedintoHostMiceinLimiEngDiluEons

Cell,Volume133,Issue4,704‐715,16May2008

CDH1=E‐cadherin

mouseHuman

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Genes involved in metastatic steps, where is the selective pressure?

Nguyen,BosandMassague,2009

Dual functions of metastasis progression genes allow for selection in the primary tumour

Nguyen,BosandMassague,2009

Promoteangiogenesis

PromoteExtravisaEon

Lysyloxidaseisinducedbyhypoxiaandpromotescancercellinvasion

LOXisECMproteininlungfacilitatespermissivenicheforcancercells

(COX2)

cytokine‐nodiscernableeffectinprimarytumour

PromotesendothelialcelldissociaEon

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Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

LinearModelofMetastasis

Inthismodel,tumourontogenyproceedstofullmalignancywithintheprimarytumourmicroenvironment,ajerwhichtumourcelldisseminaEonfoundsametastasis.Therefore,theprimarytumourprescribesthemolecularcharacterisEcsofDTCs(DisseminatedTumourCells)spreadthroughoutthebody.

ParallelModelofMetastasisInthismodel,tumourcellsdeparttheprimarylesionbeforetheacquisiEonoffullymalignantphenotypestoundergosomaEcprogressionandmetastaEcgrowthatadistantsite.TheproposiEonofearlydisseminaEonanddivergentprogressionofprimarytumoursandDTCstowardsmetastasisquesEonstheroleoftheprimarytumourfortherapypredicEon.

Two Fundamental Models of Metastasis

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Linear progression model

Supportfor:CorrelaEonwithtumoursizeandfrequencyofmetastasis.Supportagainst:averagedoublingEme157daysforbreastcancerso12yearstoreach1CM,thenanother6‐12yearsforfirstmettoreach1CM,notconsistentwithevidence.

Klein,2009

Parallel progression model

Klein,2009

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Parallel progression model-better fit with clinical data

Klein,2009

Genetic evidence supporting the Parallel progression model

SeminalpaperbySchlimokandRiethmullerdetecEngcytokeraEnposiEvecellsinbonemarrowinpaEentswithM0andM1breastcancer.Only1‐10cellsper2millionbonemarrowcells.ProcNatlAcadSciUSA.1987Dec;84(23):8672‐6

AdventofsinglecellCGHshowedbonemarrowDTCshadsignificantlyfewergeneEcaberraEonsthanprimarytumourcells.InBC50%ofcytokerraEn‐posiEvecellshadnormalkaryogramswhereasallmatchedprimarytumourkaryogramswereabnormal.ButwhileDTCwerekaryotypicallynormalltheycontainedsmalldeleEonstypicalofBCindicaEngthattheydisseminatedbeforegenome‐wideinstabilitywasacquired(Schardtetal.,2005CancerCell8:227)

ERBB2amplificaEoninDTCsdidnotcorrelatewithamplificaEoninprimarytumour.InteresEngly,ERBB2amplificaEoninprimarytumour(T1,T2)didnotcorrelatewithreducedsurvival,butpaEentsdisplayingagaininERBB2inasingleDTCfrombonemarroworlymphnodediedwithin23months.(Stoeckleinetal.,2008,CancerCell13:441)

KRASandP53mutaEonsshowheterogeneitywithinandbetweenprimarycolorectalcarcinomasandmatchedmetastases.(Albaneseetal.,2004,Biochem.Biophys.Res.Commun.325:784)

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SystemicSpreadIsanEarlyStepinBreastCancer

YvesHüsemann,JochenB.Geigl,FalkSchubert,PieroMusiani,ManfredMeyer,ElkeBurghart,GuidoForni,RolandEils,TanjaFehm,GertRiethmüllerandChristophA.Klein

CancerCell,13,58‐68,2008

A.  NumberofdetectedCK+cellsper2X106bonemarrowcellsinpaEentswithdifferenttumorstages(DCIS,n=39;T1,n=328;T2,n=202;andT3/4,n=38).TherewasnoassociaEonbetweentumorstageandthepresenceofdisseminatedcells,andspecifically,thefindingofCK+cellsinpaEentswithductalcarcinomainsitu(DCIS;13%)andT1‐stagepaEents(22%)wasstaEsEcallynotdifferent(p=0.093,Pearson'schi‐squaretest).

B.  105singleCK+cellsisolatedfrom56paEentsshowednosignificantdifferencebetweenpaEentswithsmallandlarge,indicaEngthatthewell‐knownassociaEonoflargetumorsizeanddevelopmentofmanifestmetastasisisnotexplainedbyanincreasedfrequencyofgeneEcallyprogressedcancercellsinbonemarrow

Linear and Parallel progression models-consequences for therapy

Catch‐alltherapiescanbeselectedbasedanalysisoftheprimarytumour.

Klein,2009

PredicEngresponsestotherapieswillrequirethemolecularcharacterizaEonofDTCs.

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QuesEoningmetastasisfrommetastasis

•  AcascademodelpreviouslyarguedthattargeEngthefirstmetastasismightpreventaddiEonalmetastasis.(Weissetal.,1988,JCancerRes.Clin.Oncol.114:605)

•  MedianEmefor50%ofBCpaEentswithM0diseaseandsurgerytodevelopasinglemetisthesameasformulEplemets.(KleinandHolzel,2006,CellCycle5:1788)

•  Halsted’stheoryofconEnuouscancerspreadbylymphaEcdisseminaEon(1907)isthebasisforsurgeon’sremovalof10‐12lymphnodesinpaEentswithaposiEvesenEnelnode.

•  However,threerandomizedtrialsshowednoeffectofremovalofclinicallynegaEvenodesondistantmetastasisorsurvival.(Rudenstametal.,2006,J.Clin.Oncol.24:337)

Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

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Metastasis is not random

•  Seed and soil hypothesis –  1889: Stephen Paget analyzed autopsy records of 735

women with breast cancer –  Metastasis to distant sites was not due to chance –  Certain tumor cells (the “seed”) has an affinity for the

milieu (the “soil”) of certain organs. Metastases resulted when the seed and soil were compatible

•  Regional metastases can be attributed to anatomic and mechanical factors but distant organ metastases is specific –  1964: Sugarbaker –  Lymphatic drainage to regional lymph nodes –  Organ-specific metastases: breast, prostate, and lung

cancer metastasize to the bone, while colorectal cancer metastasized to the liver and lymph nodes

Typical sites of metastatic relapse for solid tumours

Nguyen,BosandMassague,2009

• KineEcsofmetastasisisbasedonthetemporalgapbetweenorganinfiltra(onandcoloniza(onproducingaperiodofmetastaEclatency.• DramaEcallydifferentlatencyfordifferentcancertypes:Forexample,adeoncarcinomasofthebreastandlungrelapseinasimilarrangeoforgans.HoweverBCrecurrencetakeyearsordecadeswhilelungcancersestablishdistantmetastaseswithinmonthsofdiagnosis.

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Nguyen,BosandMassague,2009

Temporal course of metastasis

Dormancyorbalancedapoptosis,acquisiEonofcolonizaEonpotenEal

EarlyacquisiEonofpotenEaltoinfiltrateandcolonizeotherorgans

acquisiEonofinfiltraEonandcolonizaEonpotenEal

Organ-specific barriers to metastatic infiltration

Easiest.FenestratedtofacilitatetrafficofhematopoieEccells.Liveralso.

Harder.BasementmembraneandadjacentalveolarcellsinhibitextravisaEon.Requirespecificmediatorsoftrans‐endothelialmigraEon.

Hardest.BasementmembraneandastrocytefootprocessessinhibitextravisaEon.

Nguyen,BosandMassague,2009

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MDA-MB-231 Breast Cancer Cell Line (established as the total outgrowth of cells derived from a pleural effusion of a

patient who relapsed years after removal of the primary tumor)

Isolate Single Clonal Populations (SCPs)

Introduce Luciferase Bioluminescent Marker and GFP Fluorescence Marker

Introduce into Nude Mice by intracardiac Injection

Minn, A. J. et al. J. Clin. Invest. 2005;115:44-55

Organ Specific metastasis of Breast Cancer Cells

SCPs exhibit different abilities to metastasize to bone or lung

Bone

Lung

Minn, A. J. et al. J. Clin. Invest. 2005;115:44-55

Adrenalgland

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DisEnctorgan‐specificmetastaEcpotenEalofindividualbreastcancercellsandprimarytumors

AndyJ.Minn,YibinKang,InnaSerganova,GaoravP.Gupta,DilipD.Giri,MikhailDoubrovin,VladimirPonomarev,WilliamL.Gerald,RonaldBlasberg,andJoanMassagué

Genes that mediate metastasis to the Bone

CXCR4 – bone homing chemokine receptor CTGF – connective tissue growth factor IL-11 – activator of osteoclast differentiation (mediators of bone resorption in bone metastases) MMP1 – matrix metalloproteinase/collagenase, promotes osteolysis by cleaving a specific peptide bond in the collagen of bone matrix OPN – osteopontin (consistently overexpressed in metastatic cells)

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Metadherin,acellsurfaceproteininbreasttumorsthatmediateslungmetastasis

BrownandRuoslahE,2004,CancerCell,5,365‐374

Transmembranedomain

293cellswithectopicMetadherinexpression

Breastcancercells

Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

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Bonemarrow‐derivedcells(BMDCs)fostertumorigenesisandmetastasis

•  HaematopoieEcprogenitorcellsareimplicatedasiniEatorsofthepre‐metastaEcnicheandareinvolvedinangiogenesis.Maturemonocyteandmacrophagecellsandneutrophilssecretechemokinesandmatrix‐degradingenzymesthatmodulatethelocalmicroenvironmentandmediatethechemoawracEonofotherinflammatorycellstothepre‐metastaEcniche.

•  Endothelialprogenitorcellsaremobilizedfromthebonemarrowduringangiogenesis.IthasbeensuggestedthatrecruitmentofendothelialprogenitorcellsinsEgatesthemicrometastaEctomacrometastaEcswitch.

•  Mesenchymalstemcellsgiverisetofibroblasts,whichareimportantcomponentsofthetumourstroma.TheymayalsodirectlyinteractwithtumourcellstoenhancetheirmetastaEcphenotype

Evolution of a metastatic niche – involves a tumour-permisive immunological or inflammatory microenvironment

PsailaandLyden,2009

Beforearrivalofprimarytumorcells.ResulEngfromsystemiceffectsoffactorssecretedbyprimarytumorcells.

EPC:endothelialprogenitorcells;MSC:mesenchymalstemcells;HPC:haematopoieEcprogenitorcells;MTC:metastaEctumorcell

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Mesenchymalstemcellswithintumourstromapromotebreastcancermetastasis

AntoineE.Karnoub,AjeetaB.Dash,AnnieP.Vo,AndrewSullivan,MaryW.Brooks,GeorgeW.Bell,AndreaL.Richardson,KorneliaPolyak,RossTubo&RobertA.Weinberg

Nature2007

CCR5isexpressedbyMDA‐MB‐231cellsandnotbyMSCs.MSC‐derivedCCL5actsprimarilyinaparacrinefashionontheMDA‐MB‐231cellsintheBCCandMSCmixedcellpopulaEonstoenhancemetastasis.

Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

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AngiogenicSwitch‐HypothesisExpansionofatumormassbeyondtheiniEalmicroscopicsizeofanon‐angiogenictumorisdependentontherecruitmentofitsownvascularsupply,byangiogenesisand/orbloodvesselcoopEon.Theabilityofatumortoprogressfromanon‐angiogenictoangiogenicphenotypeiscentraltotheprogressionofcancerandistermedthe“angiogenicswitch”.

HistoryofAngiogenesisResearch

•  1970’s–HypothesisofFolkmanthattumorgrowthdependsonangiogenesis

•  1980’s‐IdenEficaEonofvasculargrowthfactors–Proofofconceptinanimalmodels

•  1990’s–ClinicalTrialsofangiogenicinhibitors–Earlyclinicalfailures‐monotherapy

•  2004‐FDAapprovalofbevacizumabformetastaEccolorectalCA

•  2007‐Bevacizumab+irinotecanefficaciousforglioblastoma

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TumorAngiogenesis

FolkmanJ,NatureDrugDiscovery6:274,2007

Figure2|Angiogenesisinratsarcoma.Inthismicrograph,bloodvesselsgrowtowardsasarcoma(darkareaatright)inratmuscle.Thiscontrastswiththenormalgrid‐likepawernofbloodvesselsthatappearsattheupperlej.(CourtesyofL.HeuserandR.Ackland,UniversityofLouisville,USA)

Escapefromtumorcelldormancyrequirestheangiogenicswitch

Duringdormancycells:(1)remainharmlesstothehostunEltheyswitchtotheangiogenicphenotype(i.e.,maybeharmlessfor1yearormore,whichishalfthelife‐spanofamouse);(2)expressequalormoreanEangiogenic(i.e.,thrombospondin‐1)comparedtoangiogenic(i.e.,VEGF,bFGF)proteins;(3)growtoapproximately1mmindiameterorlessinvivo,atwhichEmefurtherexpansionceases;(4)showacEvetumorcellproliferaEoninmice(balancedbyapoptosis),andremainmetabolicallyacEveduringthedormancyperiod.

Naumov,AkslenandJudahFolkman,2006CellCycle

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FolkmanJ,NatureDrugDiscovery6:274,2007

• AngiopoieEn1(ANGPT1)maintainsnormalbloodvessels.TumourcellssecreteANGPT2,whichcompetesforbindingtotheendothelialTIE2receptor.ANGPT2increasesthedegradaEonofvascularbasementmembraneandmigraEonofendothelialcells,thereforefacilitaEngsproutformaEon.

• Vascularendothelialgrowthfactor(VEGF)issecretedbytumourcells.Itisthemostcommonofatleastsixotherpro‐angiogenicproteinsfromtumours.OthersincludePlatelet‐derivedgrowthfactor(PDGF)andBasicfibroblastgrowthfactor(bFGF;alsoknownasFGF2).EndostaEnisanE‐mitogenic.

• Integrinsfacilitateendothelialcellbindingtoextracellularmatrixandpromotecellviability.Pro‐angiogenicproteinsupregulateendothelialintegrinstosustainendothelialcellviabilityduringtheintermiwantdetachmentsrequiredformigraEon.

• Newendothelialcellsarealsorecruitedasprecursorbone‐marrow‐derivedendothelialcells.

• Someangiogenicregulatoryproteins(bothpro‐andanE‐angiogenic)arescavengedbyplatelets,storedinalphagranulesandreleasedwithinthetumourvasculature.

Outline

•  Generalmetastasis.•  EpithelialtomesenchymaltransiEon.

•  Linearversusparallelmodelsofmetastasis.

•  Organsitespecificmetastasis.

•  ThemetastaEcniche.

•  Angiogenesis.•  AnE‐angiogenictherapy.

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CurrentAngiogenicInhibitorsinClinicalUseandClinicalTrials

•  Bevacizumab(AvasEn™)•  SuniEnib(Sutent™)•  Sorafenib(Nexavar™)•  Cederanib(RecenEn™‐AZD‐2171)•  CilengiEde•  VEGF‐TrapManyothersindevelopment

DifferentMechanismofAcEonof3FDA‐ApprovedDrugs

FolkmanJ,NatureDrugDiscovery6:274,2007

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AnEangiogenicTherapyElicitsMalignantProgressionofTumorstoIncreasedLocalInvasionandDistantMetastasis

MartaPàez‐Ribes,ElizabethAllen,JamesHudock,TakaakiTakeda,HiroakiOkuyama,FrancescViñals,MasahiroInoue,GabrieleBergers,DouglasHanahan,OriolCasanovas

AcceleratedMetastasisajerShort‐TermTreatmentwithaPotentInhibitorofTumorAngiogenesis

JohnM.L.Ebos,ChrisEnaR.Lee,WilliamCruz‐Munoz,GeorgA.Bjarnason,JamesG.Christensen,RobertS.Kerbel

Ebosetal.,2009

HumanmetastaEcbreastcancer231/LM2‐4LUC+cellswereinjectedintothetailveinofseverecombinedimmunodeficiency(SCID)miceandtreatedwithsuniEnibasindicated.

AcceleratedExperimentalMetastasisandDecreasedSurvivalajerShort‐TermSuniEnibTreatmentbeforeandajerIntravenousTumorInoculaEon

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Ebosetal.,2009

OpposingEfficaciesofShort‐termandSustainedSuniEnibTreatmentinPrimaryandMetastaEcDisease

231/LM2‐4LUC+cellswereimplantedintothemammaryfatpadofnu/numiceandtreatedwithSuniEnibasindicated.

200mm3

VEGFasanegaEveregulatorofgliomainvasion

DuRetal.,CancerCell13:206‐220,2008.

BoydenChamberinvasionassay

perivascularinvasion

HIFkohavereducedVEGFandreducedvascularizaEon,butincreasedperivasculatureinvasion,thisislikelyamechanismforthetumorcellstoevadehypoxia.

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IncreasedtumorinvasivenessandmetastasisevokedbyVEGFinhibitors

Logesetal.,2009,CancerCell

TheEnd!