癌基因与抑癌基因课件

癌基因与抑癌基因

Oncogene&TumorSuppressorGene癌生物学课程癌基因与抑癌基因

Oncogene&TumorSupp1提纲一、癌基因二、抑癌基因三、癌基因/抑癌基因与多步骤癌变四、癌基因/抑癌基因的鉴定提纲一、癌基因2肿瘤发生机理物理因子化学因子生物因子，如病毒遗传物质改变正常细胞癌细胞ageneticdiseasecharacterizedbyuncontrolledcellgrowth肿瘤发生机理物理因子化学因子生物因子，如病毒遗传物质改变正常3一、癌基因Oncogene一、癌基因Oncogene41.癌基因的发现Non-Transforming(AvianLeukemiaViruses,ALV)EllermanandBang,19081,Induceleukemiaafterlonglatencyperiods2,Donot“transform”tissueculturecellsAVirusandCancer1.癌基因的发现Non-Transforming(Avia5Transforming(SarcomaViruses,RSV)PeytonRous,1911

-cellfreelysatescouldinducesarcomasinotherchickens1，Acute;2，Transformculturedcells病毒致癌--NobelPrize!1966BTransforming(SarcomaViruses,6IsolationofRetroviralTransformingGenesRSV(gag,pol,env,src)R.T.(1975Nobel)gag,polenvsrccDNARSV(td:gag,pol,env)gag,pol,envDenatureandHybridize1975,1977unhybridizedsequenceshybridizedsequencesgag,pol,envsrcgenomicRNACIsolationofRetroviralTransf7probeRSV-InfectedCEF(+control)“Normal”chickDNAMouseDrosophilaHuman+++++1989NobelPrizeforBishopandVarmusThus:aproto-oncogeneistheNORMALprogenitorgeneofaviraloncogeneCont.1980,1stoncogeneidentifiedSrc（v-onc,viraloncogene）probeRSV-Infected“Normal”Mou8癌基因与抑癌基因课件9HumanBladderTumorcelllineDNAIsolatehighMWDNAIsolateDNAfragmentsRestrictionendonucleaseTransfectionNIH3T3fibroblastsTransformationIsolateDNA(>99%mouse+8-10humangenes)TransformationIdentifyHumanDNAActivatedProto-oncogenesfromDNAtransfectionDHumanBladderTumorIsolatehig10RESULT:RAS“Activation”isduetoaSINGLEpointmutation(glyval)atcodon12UseAluprobeIsolateHumanDNARESULT:ASINGLEhumangeneisresponsiblefortransformingcapabilitySequencingRESULT:ThegeneistheHUMANc-H-rasgeneComparesequencetoNORMALgene1sthumanoncogenemutationRasG12Vwasidentified!1982,RobertWeinberg,etcthreegroupsCont.RESULT:RAS“Activation”UseA112.癌基因定义DefinitionViralOncogene(v-onc):

agenecarriedbyatumorvirus(RNAorDNA),theexpressionofwhichisnecessaryandsufficient

toinducetransformationintissueculturecellsandtumorsintheappropriateanimal.V-oncisencodedbycellularsequencesthathavebecomeinsertedintotheviralgenome.Oncogene:analteredgenewhoseproductcanactinadominantfashiontohelpmakeacellcancerous.Typically,anoncogeneisamutantformofanormalgene(proto-oncogene)involvedinthecontrolofcellgrowthordivision.Proto-oncogene(c-onc):anormalcellularformofagenethatcontrolscellproliferationandcanbeconvertedintoacancer-promotinggenebymutation,whosecontinuedactivationleadstocontinuedsignaltransduction,andwhoseaberrantexpressionoractivitymaycontributetotumorigenesis.2.癌基因定义DefinitionViralOncoge12原癌基因特点1．Controlscellproliferationandsurvival;2．Canbeconvertedintooncogene,andinducetransformation3．Conservedacrossorganisms4．Tissue-specificPhysiologicalfunction:cellsignalingpathwaystightlycontrolled“Hyper”-functional原癌基因特点1．Controlscellprolifer133.原癌基因激活机制TransductionviaretrovirusesLTRLTRViralRNAPackagingOfretrovirusgagpolenvProto-oncogeneis“captured”or“usurped”fromhostcellgenomeRetroviruswithoutoncogeneLTRLTRgagpolenvSrcRetroviruswithoncogeneA3.原癌基因激活机制Transductionviaret14Retroviralpromoter/enhancerinsertionBRetroviralpromoter/enhanceri15

ChronicMyelogenousLeukemia(CML)：chromosome9q34(c-abl)

chromosom22q11.2(bcr)，proto-oncogenec-ablisactivatedChromosomaltranslocationq3495’3’5’3’22q11.2ablder9Ph’abl9q3422q11.222q11.29q34Der(22)DNARNAProteinbcrbcrbcr/ablFusionproteinCChronicMyelogenousLeukemia16

Gainofmultiplecopiesofdefinedchromosomalregions(1)Homogeneouslystainingregion(HSR)

(2)Doubleminutechromosomes(DM)TumorOncogeneAmplificationSmallcelllungcancerc-mycUpto8OXN-mycUpto50XL-mycUpto20XNeuroblastomasN-mycUpto250XGlioblastomasc-erbB1(EGFR)Upto50XMammarycarcinomac-erbB2(HER2)Upto30Xc-mycUpto50XCyclinD1Upto30X

AmplificationCellularProto-oncogenesamplifiedinhumantumorsDHSRDMGainofmultiplecopies17Ras：P21transformingprotein

c-H-ras---Harveyratsarcomavirusc-K-ras---KirstenratsarcomavirusN-ras---Neuroblastoma

PointmutationHotmutationpoints:12,13,6112Gly---Valwt-RasGTPwt-RasGDPActiveformInactiveformmt-RasGTPmt-RasGDPConstitutivelyactiveERas：P21transformingprotein

P18growthfactors(sis)GTPaseproteins(ras)guaninenucleotideExchangeproteins(rho)CytoplasmicMembraneassociatedTyrosinekinases(src)growthfactorreceptors(erb-b)nucleartranscriptionfactors(myc)cytoplasmicserineThreoninekinases(akt)Accordingtolocationandfunctionofproto-oncogeneproducts4.癌基因分类cAMPDGIP3Ca2+growthfactorsGTPaseproteinsg19癌基因与抑癌基因课件20Overview:Classesofoncogenes

A.SecretedGrowthFactors(e.g.SIS,TGF-α,etc)>>inducecellgrowthbymobilisationofenergystores,differentiationandentryintothecellcycle.B.Receptors(cellsurface)

>>differentcellshavedifferentreceptors,therebyasignalcanproducearesponseinsomecelltypebutnotothers

Cellsurfacereceptorwithproteintyrosinekinase(PTK)activity(erbB,neu/erbB-2,ros,fms)C.IntracellularTransducers>>actassecondmessengerswhichaltertranscription,eitherbyallowingnewgenestobeexpressedorbymodifyinglevelsofexpressionofalreadyactivegenes

a-ProteinTyrosineKinase(src,yes,fps,abl,met) b-Protein-Serine/Threoninekinases(akt,

mos,raf) c-Rasproteins(Ha-ras,Ki-ras,N-ras)

d-Adaptors(crk)D.NuclearTranscriptionFactors>>specificbindingproteinsthatrecogniseshortsequencemotifswithinthepromotersandenhancers.ThesefactorsthenaccelerateorretardtherateofinitiationoftranscriptsbyRNApolymeraseII a-jun,fos

b-myc,N-myc,myb,ski,relOverview:Classesofoncogenes21BasicCellularSignalingMachinery:forexampleKinasesignalingandCancerReceptorProteinTyrosineKinaseSignalingNon-receptorProteinTyrosineKinaseSignaling

IntracellularSerine/ThreonineKinaseSignaling5.癌基因功能ABCGTPaseproteinsDNucleartranscriptionfactorEBasicCellularSignalingMachi22GrowthFactorReceptorstrans-membrane(glyco)proteinspossessintrinsicproteintyrosinekinase(PTK)activitydimerize(homoandheterodimers)auto-,trans-phosphorylationrecruitsignalingmoleculesatspecifiedphosphorylatedsitesAberrantlyexpressedinmanytumorsGFGFPPPPPGFPPPPReceptorProteinTyrosineKinaseSignalingAGrowthFactorReceptorstrans-m23CollectionofReceptorproteinTyrosineKinases(RPTK/RTK)CollectionofReceptorprotein24GROWTHFACTORRECEPTORTYROSINEKINASEGRB-2SOSRASRAFMAPKKMEMBRANEMAPKDimerization;autophosphorylationInteractionofsignalingmoleculesActivationof“downstream”kinasesJAKSTATPNUCLEUSMAPKPhosphorylationoftranscriptionfactorsSTATGENEEXPRESSION,i.e.,fos,jun,mycCYTOPLASMPPPPPPPPPPPPPPPPPPPPPRPTKSIGNALINGCASCADES25GROWTHFACTORRECEPTORGRB-2SOSR25PTK(Proto-oncogene)Viraloncogene*(viraloncoprotein)OncogenicalterationTumour/cancertypes(onlythemostfrequent,Mainlyhumantypesaredescribed)EGFR/ErbB1(c-erbB)V-erbBfromAEV(p68/74abB)V-erbB:TruncatedEGFRPTKC-erb:Overexpression(amplification)Extracellulardomaindeletionsv-ErbB:fibrosarcomasc-ErbB:mammarycarcinoma,glioblastomamultiforme,Cvarian,non-small-celllungandlthercancersErbB2/HER2/NeuOverexpression(amplification)Norecurrenthumanmutations(Val664gluinrodents)Mammary,ovarian,gastric,non-small-celllungandcoloncancerErbB3/HER3Overexpression;constitutivetyrosinephosphoylation(heterodimerwithErbB2)MammarycarcinomaErbB4/HER4OverexpressionMammarycarcinoma,granulasacelltumoursIGF-1ROverexpression(expressionrequiredforinvitrotransformationbymanyoncogenesandDNAviruses)Cervicalandothercarcinomas,sarcomasPDGFR-

Overexpression(amplification)Glioma,glioblastoma,ovariancarcinomaPDGFR-

Tel-PDGR-(t(5;12)translocationfusingEts-likeTelwithPDGFR-PTKdomain)OverexpressionTel-PDGFR-:chronicmyelomonocyticleukaemiaPDGFR-:gliomaCSF-1R(c-fms)V-fmsfromFesV(p170gag-fms)v-fms:TruncatedCSF-1RPTKwithmutantC-terminaltailConstitutivelyactivec-fms:GOFpointmutationsOverexpressionv-fms:jelinesarcomasc-fims:acuteandchronicmyelomonocyticleukaemias,monocytictumours,malignanthistiocytosis,endometrialcancer,gliomaKit/SCFR(c-kit)V-kitfromFeSV(p80gag-kit)v-kit:TruncatedKit/SCFRPTKwithmutantC-terminaltailConstitutivelyactivec-kit:GOFpointmutationsandsmalldeletionsOverexpressionV-kit:felinefibrosarcomasC-kit:malignantgastrointestinalstromaltumours,acutemyeloidleukaemias,myelodysplasticsyndromes,mast-cellleukaemia/systemicmastocytosis,seminomas/dysgerminomas,small-celllungcancerandothercarcinomasReceptorProteinTyrosineKinasesandCancerPTKViraloncogene*Oncogenical26actinCIP4DNAFABDFERMkinaseKinase-likePHSH2SH3Actin-bindingdomainBtkmotifCdc42-bindingCIP4homologydomainDNA-bingingdomainFocaladhosion-bindingdomainIntogcyin-bindingdomainPIKdomainPscodoPIKdomainPlockstrinhomologydomainSrchomology-2domainSichomology-3domainSH2kinaseSH3DNAactinSH2kinaseSH3FERMKinase-likekinasekinaseSH3SH3SH2kinasekinaseFERMFABDkinaseCIP4SH2SH3kinaseSH2SH3SH2kinaseSH2SH2kinasePHSRCABLJAKACKCSKFAKFESFRKTECSYKFGR,FYN,SRC,YES1,BLK,HCK,LCK,LYNBAL,ARGJAK1,JAK2,JAK3,TYK2ACK1,ACK2CSK,MATK/CTKFAK,FESFA,PYK2BRK,FRK,SRMSBMX,BTK,ITK,TEC,TXXSYK,ZAP70Non-receptorProteinTyrosineKinaseSignaling

BactinCIP4DNAFABDFERMkinaseKina27PTK(Proto-oncogene)Viraloncogene*(viraloncoprotein)OncogenicalterationTumour/cancertypes(onlythemostfrequent,Mainlyhumantypesaredescribed)ROS(c-ros)v-rosfromavianUR2SV(p68gag-ros)v-ros:TruncatedRosPTKdomain.Constitutivelyactivec-ros:OverexpressionRaretruncations/pointmutations?v-ros:avianfibrosarcomasC-ros:glioblastomas,astrocytomasAlkNPM-Alk(t(2;5)nucleophosminfusedtoAlkPTKdomain)lg-Alk(t(2;22)lgfusedtoAlkPTKdomain)Othersporadicfusionswithtropomyosin,etc.Non-Hodgkinlymphomas,CD30+andCD30-anaplasticlarge-celllymphomaSrc(c-src)v-srcfromRSV(pp60v-src)v-src:C-teminaltruncationandpointmutations(increasedkinaseactivity)c-src:C-terminaltruncation(increasedkinaseactivity)Overexpressionand/orincreasedkinaseactivitypp60v-sre:aviansarcomasc-Srctruncation:coloncancerc-Srcoverexpression:mammaryandpancreaticcancers.neuroblastomas,othersAbl(c-abl)V-ablfromAbelsonMLV(p160gag-abl)orfromP1-FeSVv-abl:N-terminal(SH3)truncationot

AblFusions:p190Bcr-Abl,(t(9;22)m-bcr);p210bct-Abl,(t(9;22)M-bcr);p230bcr-Abl,(t(9;22)-bcr).M-,m-and-bcr:3breakpointclusterregionsinBCR.ThechimaericmRNAusuallystartswithexona2ofABL,itneverincludesexon1aor1b.Tel-Abl(t(12;22)N-terminal(H-L-HregionofTelfusedwithAblexon2ap160gag-abl:murineacuteleukaemiasp190Bct-Abl:~50%ofPh+acutelymphocyticleukaemias,rarelychronicmyelomonocytic

leukaemiasp210Bcr-Abl:chronicmyeloidleukaemias,~30%ofPh+acutelymphocyticleukaemiasp230Bcr-Abl:somePh+chronicneutrophilic

leukaemiasTel-Abl:rarecasesofacutelymphocyticleukaemiasNon-receptorProteinTyrosineKinasesandCancerPTKViraloncogene*Oncogenical28SrcStructureSrcStructure29BasalActivty(discernedfromcrystalstructure)ActivatedKinaseSrcdomainsandSrcactivityBasalActivty(discernedfrom30PI-3-kinaseSrcFamilyrasmycmitosismitoticfunctionsstresspathwaysextracellularmatrixcytoskeletalreorganizationantigensoxidativestresscytokinesGproteincoupledreceptorsRPTKsangiogenesisTheWorldAccordingtoSrcPI-3-kinaseSrcrasmycmitosismit31RASsuperfamlyCGTPaseproteins1.H-ras,chr11;K-ras,chr12;N-ras,chr12.5exons,188-189aa,MW21KD3.GTPaseRASsuperfamlyCGTPaseproteins32RASsignalingRASPI3KPIP3Akt/PKBRho-GEFRaf(MAPKKK)MEK(MAPKK)Erk1/2(MAPK)Mnk1,RSK,Ets,Elk-1,SAP-1Ral-GEFRal-ARal-BCdc42,RacRASsignalingRASPI3KPIP3Akt/P33TheWorldAccordingtoRASTheWorldAccordingtoRAS34RASmutationandCancerRASmutationandCancer35CatalyticregPHT308*S473*LipidbindingSer/thrkinase*FullactivationofkinaserequiresphosphorylationofbothT308andS473AktstructureIntracellularSerine/ThreonineKinaseSignalingDCatalyticregPHT308*S473*Lipid36CatalyticregPHT308S473PI3KactivationPI(3,4,5,)P3PDK1PPPDK2,PDK1,ILKCatalyticregPHT308S473PPPI(3,4,5,)P3NUCLEUSGrowthfactorreceptorsPI(4,5,)P2AktactivationCatalyticregPHT308S473PI3KPI(337CatalyticregPHT308S473GSK3PFK-2PDE-3BmTORIkBBadp21ForkheadGlycogensynthesisProteinsynthglycolysiscAMPtranslationExpressionofFasligandExpressionOfantiapoptoticgenesBcl-XLBcl-2CellcycleAkt/PKB-mediatedsignalsCatalyticregPHT308S473GSK3PFK-38PI3KandMEKpathwayincancerPI3KandMEKpathwayincancer391.c-myc,8q24,439aa;N-myc,2p23-24,456aa;L-myc,1p32,364aa2.NucleartranscriptionfactorNucleartranscriptionfactorEMyc1.c-myc,8q24,439aa;N-myc,40MYCregulationMYCregulation41癌基因与抑癌基因课件42二、抑癌基因TumorSuppressorGene二、抑癌基因43EvidencesforTumorSuppressorGenesSomaticCellGeneticStudies1969,EphrussiandHarrisRasoncogene-NIH3T3-transformationRasoncogene-CHO-NOtransformationDNAtransfection1.抑癌基因的发现EvidencesforTumorSuppressor44PEDIGREEofafamilywithfamilialretinoblastomawaspublishedbyThaddeusP.Dryjaandhiscollaborators.Affectedmembersareindicatedbysolidcircles(females)orsquares(males).Fivechildreninthesecondgenerationdevelopedthetumor.Onesonwhowasunaffectedhadnonethelessinheritedamutatedchromosome13:twoofhisdaughterswereaffected.13q14CytogeneticAnalysisoffamilialcancersPEDIGREEofafamilywithfami45CloningandIdentificationofRBgene1986CloningofRbgenePutRbgenebackintoRbcells--losetransformingabilitysuchastumorigenicity,softagarcolonyformingability1sttumorsuppressorgeneidentified!CloningandIdentificationof46Terminology:Tumorsuppressorgenes,Anti-oncogenes,RecessiveoncogenesTumorSuppressorGene：agenewhoseproductcannegativelycontrolcellgrowthandsuppresscelltransformation.1.Negativelycontrolcellgrowthanddivision2.Suppresscelltransformation3.Mutatedandinactivatedincancer4.tissue-specificCriteria:A.ExpressesinnormaltissuesB.Mutatedincancer：DNA,mRNA,proteinC.Re-introductionmakecancercelllosetransformingabilityFeatures:2.抑癌基因定义Terminology:Tumorsuppressor47Knudson’sTwo-hittheoryDefinition:inactivationofoneallelebymutationsorsmalldeletionsandlossofthesecondallele,usuallybychromosomeloss(lossofheterozygosity).-chromosomeloss-mutations-smallintragenicdeletions-promotermethylation(epigenetics)HaploinsufficiecyInactivationofonealleleisenoughtoleadtotumorgrowth3.抑癌基因失活机制Epigenticderegulation

ThemethylationstatesofpromoterandhistoneaffecttheexpressionlevelofgeneKnudson’sTwo-hittheoryDefini481sthit2ndhitmutMemutmutMeMeMeMeMutationMethylationLossMethylationLossMethylationMutationMutationMethylationMethylation

++++

LOHmethylationChromosomelossMethylation1sthit2ndhitmutMemutmutMeMeM49癌基因与抑癌基因课件50CancersyndromeGenePrincipalTumorsProteinProductLocalizationModeofActionRetinoblastomaRB1Retinoblastoma.OsteosarcomaNucleusTranscriptonalregulator/factorLi-Fraumenip53Sarcomas.breastandbraintumorsNucleusTranscriptonfactorFamilialadenomatouspolyposisAPCAdenomatouspolyps.coloncancerCytoplasmRegulates

-cateninfunctionWilmstumorWT-1NephroblastomaNucleusTranseriptionfactorNeuofibromatosistype1NF-1Neurofibromas.sarcomas.GliomasCytoplasmP21ras-GTPaseactivatorFamilialmelanomaandpancreaticcancerp16Melanoma,pancreaticcancer.NucleusCyclin-dependentkinaseinhibitorFamilialbreastcancerBRCA1BRCA2BreastandovariancancerBreastNucleusUnknownDNArepair,chromosomalstabilityTumorSuppressorGenesAssociatedWithaCancerSyndromeCancersyndromeGenePrincipalT51Receptor：PTCHDCCIntermediateregulators：NF1PTENAPCNF2Transcriptionfactors/activators：p53WT1RB1VHLCellcycleinhibitors：p16p21p15DNArepair：MSH2MLH1PMS2ATMBRCA1BRCA2Accordingtolocalizationandmolecularfunction4.抑癌基因分类Receptor：PTCHDCCAccording52Gatekeepersvs.CaretakersGatekeepers–Genesdirectlyregulatingtumorgrowthbyinhibitingtheirgrowthorpromotingtheirdeath.

Ex.PTEN,p53,RBCaretakers–Genesthatdonotdirectlysuppresstumorgrowth,i.e.,inactivationofthesegenesleadstogeneticinstabilitythatindirectlypromotestumorgrowth.

Ex.BRCA1,BRCA25.抑癌基因功能Gatekeepersvs.CaretakersGate531.17p13.12.11exons,393aa,MW53KD3.Transactivator(TA)TranscriptionalActivationdomainDNAbindingdomainTetramerizationdomainGeneandproteinstructureTP531.17p13.1TranscriptionalDNA54Evidences:Mutationsincancers:LossofHeterozygosityColorectalcancersInhibitionoftransformationAp53nullmouse

viablelossofoneorbothp53allelesgivesatumorphenotypelossofp53cooperateswithotheralterationstoincreasetumorincidencep53:Li-FraumeniSyndrome-aninheritedpredispositiontocancer-variouskindsofcancersoccur:osteosarcomas,softtissuesarcomas,etc-multipletumorsinthesameindividualAtumorsuppressorgeneEvidences:Mutationsincancers55Classificationofp53mutations1.Loss-of-function2.Dominant-negativeMutant:lossoffunctionWidetype:normal3.Gain-of-functionMutant:lossoffunctionWidetype:lossoffunctionp53p53p53p53TranscriptionalactivationMutantp53p53p53p53BindsandregulatestranscriptionofothergenesMDR-1,c-mycClassificationofp53mutation56p53functionsTranscriptionalActivator:bindstocis-element

Consensussequence:P：G/APy：C/T

ATPPPCGPyPyPyTACellcycleregulatorygenes

p21/WAF1/Cip1,GADD45,cyclinGApoptosisgenes

Bax,Puma,Noxa,

etcRegulatorsofitself

Mdm2,ARFp53functionsTranscriptionalA57Inactivationofp53UbiquitinationanddegradationbyMdm2(E3ligase)p53mdm2p53E2E1p53UBUBmdm2Interactionwithvirusproteins:SV40largeTAg,HPV16E6,E1BTAgDNAbindingDominant-negativeMutantp53Inactivationofp53Ubiquitinat58Activationofp53:increaseofstabilityInhibitionofMdm2p53Mdm2p19ArfPhosphorylationofp53UltravioletlightIonizingradiationDrugs(cisplatin,adriamycin)

ATMATRChk1Chk2DNA-PKp53pppppmdm2outAcetylationofp53Acetyltransferase:p300/CBP,PCAF,etc

Sumoylationofp53(Smallubiquitinrelatedmolecules)p53asActivationofp53:increaseof59Thep53signalingnetworkThep53signalingnetwork60癌基因与抑癌基因课件61PTEN

(phosphataseandtensinhomologdeletedonchromosome10,PTEN);MMAC1(mutatedinmultipleadvancedcancers1);TEP1(TGF-regulatedandepithelialcell-enrichedphosphatase1)GenestructurePTEN/MMAC1,

discoveredin1997PTEN(phosphataseandtensin62RegulationofPTENA.ConstitutivePhosphorylation(bycaseinkinase2)C.Ubiquitination(proteasome

degradation)B.Lipidinteraction(PIP2)(Membranelocalization)D.Proteininteractions(stability,etc)RegulationofPTENA.Constitut63PI–3.4.5P3

PI-4.5P2PTENPI3KPTENactivityPI–3.4.5P364CanonicalPTEN–PI3K–AKT–mTORpathwayCanonicalPTEN–PI3K–AKT–mTORp65BiologicalfunctionsofPTENintumorigenesisPTENAktForkheadp27Down-regulationGSK-3ßcyclinD1degradation2.CellcyclearrestinG1phaseRac,RhoMMP-23.InhibitsmigrationandinvasionmigrationinvasionHIF-1αVEGFangiogenesis4.InhibitsangiogenesismTORelF4Edisfunction1.proteinsynthesisinhibitionBiologicalfunctionsofPTENi66PTENincancerPTENincancer67Cont.Cont.68SchematicofRBincellcyclecontrol.SchematicofRBincellcycle69AlterationsoftheCdK–RB–E2FpathwayinhumancancerAlterationsoftheCdK–RB–E2F70癌基因与抑癌基因课件71三、癌基因/抑癌基因与多步骤癌变SynergyofOncogenes&TumorSuppressorGenesinMulti-stepTumorigenesis

三、癌基因/抑癌基因与多步骤癌变721.Multi-hitrequirementsforcarcinogenesisNIH3T3transfectedassayisanone-hitmechanism(Rastransformation)Epidemiologysupportsmulti-hitrequirementsformosttumorsOnly20%ofhumantumorDNAproducefociinNIH3T3transfectionassayProblems: NIH3T3isaimmortalcellline.

NIH3T3cellisnotexactlynormal!

Howaboutfoci-formingassaysusingembryofibroblasts?1.Multi-hitrequirementsfor73TransfectionofRatDiploidEmbryoFibroblastsREFNOFOCI!!!RasGeneREFNOFOCI!!!v-mycGene+RasFOCICONCLUSION:Atleasttwogenesarerequiredfortransformationof“normal”cellsTransfectionofRatDiploidEm74ComplementationgroupsofProto-oncogenesESTABLISHMENT(IMMORTALIZATION)GENESc-myc,N-mycE1A(adenovirus)LargeT(polyoma)NUCLEARTRANSFORMINGGENESc-K-ras,C-H-rasN-rasc-neuCYTOPLASMICComplementationgroupsofProt75Tumorformationcanbemimickedinthelab.Bydeliveryofthefollowinggenes:CatalytichTERTsubunitoftelomerase:Maintainstelomerelength2. SV40largeTantigen:inactivatesboththep53andRbtumorsuppressorgeneActivatedoncogenicrasoncogene:inducestransformationtoacancerousstate allowingcellstogrowindefinitelyintheabsenceof growthfactorsDisruptionoftheintracelluarpathwaysregulatedbythelarge-T,oncogenicrasandtelomerasesufficetocreateahumantumorcell.CreationofhumantumorcellswithdefinedgeneticelementsHahn,W.C.,Counter,C.M.,Lundberg,A.S.,Beijersbergen,R.L.,Brooks,M.W.,andWeinberg,R.A.Nature400,464-468,1999Tumorformationcanbemimicke761.WhiteheadInstituteforBiomedicalResearch2.DepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,Massachusetts02142CancerCell,Vol.6,171-183,August2004AnnapoorniRangarajan,1SueJ.Hong,2AnnieGifford,1andRobertA.Weinberg1,2,*Species-andcelltype-specificrequirementsforcellulartransformation1.WhiteheadInstituteforBio77TheLongRoadtoCancerInitiationPromotionProgressionInvasionandMetastasis2.SynergyofMulti-hitsinMulti-stepHumanCarcinogenesisTheLongRoadtoCancerInitiat78ModelforColonCancerDevelopmentandProgressionAfterVogelsteinandcolleaguesAPCModelforColonCancerDevelop793.BeyondOncogenesandTumorSuppressorGenesCell,Vol.100,57–70,January7,2000TheHallmarksofCancerDouglasHanahan*andRobertA.Weinberg†Cell,Vol.144,646–674,January7,2011DouglasHanahan*andRobertA.Weinberg†3.BeyondOncogenesandTumorS80

四、癌基因/抑癌基因的鉴定IdentificationofOncogene/TumorSuppressorGenes四、癌基因/抑癌基因的鉴定81Cancer:adiseasefromgenomeonCancer:adiseasefromgenome82HCCisthemostcommonprimarylivermalignancyWorldwideincidence>600,000casesperyearMorecommoninmenthanwomen(4:1)Forresection,rateofrecurrencecanbeashighas50%at2yearsOnly12%areeligibleforresectionorfortransplant80%-90%ofHCCcasesoccurincirrhoticliversInternationalAgencyforCancerResearch.Globocan2002.Availableat:http://www-dep.iarc.fr.AccessedFebruary19,2008;ParkinDMetal.IntJCancer.2001;94;153-156;AmericanCancerSociety.CancerFacts&Figures2007.Atlanta,GA;AmericanCancerSociety,2007.McGlynnKAetal.IntJCancer.2001;94:290-296;McGlynnKAetal.CancerEpidemiolBiomarkersPrev.2006;15:1198-1203;JemalAetal.CACancerJClin.2006;56:106-130;El-SeragHB.Gastroenterology.2004;127:S27-S34.HCCEpidemiologyHCCisthemostcommonprimary83Singlebase-pairchangesPointmutations(1per800bp)Smallinsertions/deletionsFrame-shift,microsatellite,mini-satelliteMobileelementsRetro-elementinsertions(300bp-10kb)Large-scalegenomiccopynumbervariation(>10kb)Large-scaleDeletionsSegmentalDuplicationsLocalRearrangementsChromosomalvariationTranslocation,inversion,fusionCytogeneticsNucleotideUnderstandingCancergenomicsSinglebase-pairchangesCytoge84CytogenetictechniquesHybridization(CGH,FISH)arrayCGHSNParraysNext-generationsequencing(NGS)PCRbasedre-sequencingTP53,EGFR,KRASmutationsincancersPhiladelphiachrominCMLCNV,fusiongenesCNV,fusiongenesSNV,Indels,CNV,fusiongenesGWAS,CNVTargetre-sequencingApanelofgenemutationsincancersTechniquesappliedincancergenomicsCytogenetictechniquesHybridiz85Schematicofthecontributionsofmajorriskfactors(HCC).AnnuRev

Genomics

HumGenet.

2012;13:171-205.Schematicofthecontributions86Currentprogress：Next-generationsequencing(NGS)ofHCCgenomeCurrentprogress：Next-generati87Currentprogress：Next-generationsequencing(NGS)ofHCCgenomeCurrentprogress：Next-generati88NatGenet.2011May;43(5):464-9.Whole-genomesequencingofHCV-positiveHCCgenomeNatGenet.2011May;43(5):464-89CharacterizationofrearrangementsinlivercancerWhole-genome