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4/6/09
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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!