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1、癌基因与抑癌基因癌基因与抑癌基因Oncogene&Tumor Suppressor Gene癌生物学课程提提 纲纲一、一、癌基因癌基因二、二、抑癌基因抑癌基因三、三、癌基因癌基因/抑癌基因与多步骤抑癌基因与多步骤癌变癌变四、四、癌基因癌基因/抑癌基因的抑癌基因的鉴定鉴定肿瘤发生机理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遗传物质改变正常细胞正常细胞癌细胞癌细胞 a genetic disease characterized by uncontrolled cell growth一、一、癌基因癌基因 Oncogene1.癌基因的发现癌基因的发现Non-Transforming(A
2、vian Leukemia Viruses,ALV)Ellerman and Bang,1908Ellerman and Bang,19081,Induce leukemia after long latency periods2,Do not“transform”tissue culture cellsAVirus and CancerTransforming(Sarcoma Viruses,RSV)Peyton Rous,1911Peyton Rous,1911 -cell free lysates could induce sarcomas in other chickens1，Acut
3、e;2，Transform cultured cells病毒致癌病毒致癌-Nobel Prize!1966BIsolation of Retroviral Transforming GenesIsolation of Retroviral Transforming GenesRSV(gag,pol,env,src)R.T.(1975 Nobel)gag,pol envsrccDNARSV(td:gag,pol,env)gag,pol,envDenature and Hybridize1975,1977unhybridizedsequenceshybridizedsequencesgag,pol
4、,envsrcgenomic RNACprobeRSV-InfectedCEF(+control)“Normal”chick DNA MouseDrosophilaHuman+1989 Nobel Prize for Bishop and VarmusThus:a proto-oncogene is the NORMAL progenitor gene of a viral oncogeneCont.Cont.1980,1st oncogene identifiedSrc（v-onc,viral oncogene）Human Bladder Tumor cell line DNAIsolate
5、 high MW DNAIsolate DNA fragmentsRestriction endonucleaseTransfectionNIH 3T3fibroblastsTransformationIsolate DNA(99%mouse+8-10 human genes)TransformationIdentify Human DNAActivated Proto-oncogenes from DNA transfectionDRESULT:RAS“Activation”is due to a SINGLE point mutation(gly val)at codon 12Use Al
6、u probeIsolate Human DNARESULT:A SINGLE human gene is responsible for transforming capabilitySequencingRESULT:The gene is the HUMAN c-H-ras geneCompare sequenceto NORMAL gene1st human oncogene mutationRas G12V was identified!1982,RobertWeinberg,etcthreegroupsCont.Cont.2.癌基因定义癌基因定义 DefinitionViral Vi
7、ral Oncogene Oncogene(v-onc):(v-onc):a gene carried by a tumor virus(RNA or DNA),the expression of which is necessary and sufficient to induce transformation in tissue culture cells and tumors in the appropriate animal.V-onc is encoded by cellular sequences that have become inserted into the viral g
8、enome.Oncogene:Oncogene:an altered gene whose product can act in a dominant fashion to help make a cell cancerous.Typically,an oncogene is a mutant form of a normal gene(proto-oncogene)involved in the control of cell growth or division.Proto-oncogene Proto-oncogene(c-onc):(c-onc):a normal cellular f
9、orm of a gene that controls cell proliferation and can be converted into a cancer-promoting gene by mutation,whose continued activation leads to continued signal transduction,and whose aberrant expression or activity may contribute to tumorigenesis.原癌基因特点原癌基因特点1 1Controls cell proliferation and surv
10、ival;Controls cell proliferation and survival;2 2Can be converted into oncogene,and induce transformationCan be converted into oncogene,and induce transformation3Conserved across organisms4Tissue-specificPhysiologicalfunction:cellsignalingpathwaystightlycontrolled“Hyper”-functional3.原癌基因激活机制原癌基因激活机制
11、Transduction via retrovirusesLTRLTRLTRLTRViral RNAPackagingOf retrovirusgag pol envgag pol envProto-oncogene is“captured”or Proto-oncogene is“captured”or“usurped”from host cell genome“usurped”from host cell genomeRetrovirus without oncogeneRetrovirus without oncogeneLTRLTRLTRLTRgag pol env gag pol e
12、nv SrcSrcRetrovirus with oncogeneRetrovirus with oncogeneARetroviral promoter/enhancer insertionB Chronic Myelogenous Leukemia(CML)Chronic Myelogenous Leukemia(CML)：chromosome 9q34(c-abl)chromosome 9q34(c-abl)chromosom22q11.2(bcr)chromosom22q11.2(bcr)，proto-oncogene c-abl is activatedproto-oncogene
13、c-abl is activatedChromosomal translocationq349535322q11.2ablder 9Phabl9q3422q11.222q11.29q34Der(22)DNA RNA Proteinbcrbcrbcr/abl Fusion proteinC Gain of multiple copies of defined chromosomal regionsGain of multiple copies of defined chromosomal regions (1)Homogeneously staining region(HSR)(1)Homoge
14、neously staining region(HSR)(2)Double minute chromosomes 2)Double minute chromosomes (DM)(DM)Tumor OncogeneAmplificationSmall cell lung cancer c-myc Up to 8OX N-myc Up to 50X L-myc Up to 20XNeuroblastomas N-myc Up to 250XGlioblastomas c-erbB1(EGFR)Up to 50XMammary carcinoma c-erbB2(HER2)Up to 30X c-
15、myc Up to 50X Cyclin D1 Up to 30X AmplificationCellular Proto-oncogenes amplified in human tumorsDHSRDMRasRas：P21 transforming proteinP21 transforming protein c-H-ras-Harvey rat sarcoma virus c-H-ras-Harvey rat sarcoma virus c-K-ras-Kirsten rat sarcoma virus c-K-ras-Kirsten rat sarcoma virus N-ras-N
16、euroblastoma N-ras-NeuroblastomaPoint mutationHot mutation points:12,13,61 12 Gly-Valwt-RasGTPwt-RasGDPActive formInactive formmt-RasGTPmt-RasGDPConstitutively activeEgrowth factors(sis)GTPase proteins(ras)guanine nucleotideExchange proteins(rho)Cytoplasmic Membrane associated Tyrosine kinases(src)g
17、rowth factor receptors(erb-b)nuclear transcription factors(myc)cytoplasmic serineThreonine kinases(akt)According to location and function of proto-oncogene products4.癌基因分类癌基因分类cAMP DG IP3 Ca2+Overview:Classes of oncogenes A.Secreted Growth Factors(e.g.SIS,TGF-,etc)induce cell growth by mobilisation
18、of energy stores,differentiation and entry into the cell cycle.B.Receptors(cell surface)different cells have different receptors,thereby a signal can produce a response in some cell type but not others Cell surface receptor with protein tyrosine kinase(PTK)activity(erbB,neu/erbB-2,ros,fms)C.Intracel
19、lular Transducers act as second messengers which alter transcription,either by allowing new genes to be expressed or by modifying levels of expression of already active genesa-ProteinTyrosineKinase(src,yes,fps,abl,met)b-Protein-Serine/Threoninekinases(akt,mos,raf)c-Rasproteins(Ha-ras,Ki-ras,N-ras)d-
20、Adaptors(crk)D.Nuclear Transcription Factors specific binding proteins that recognise short sequence motifs within the promoters and enhancers.These factors then accelerate or retard the rate of initiation of transcripts by RNA polymerase IIa-jun,fos b-myc,N-myc,myb,ski,relBasic Cellular Signaling M
21、achinery:for example Kinase signaling and CancerReceptor Protein Tyrosine Kinase Signaling Non-receptor Protein Tyrosine Kinase SignalingIntracellular Serine/Threonine Kinase Signaling5.癌基因功能癌基因功能ABCGTPase proteinsDNuclear transcription factorEGrowth Factor Receptorstrans-membrane(glyco)proteinsposs
22、ess intrinsic protein tyrosine kinase (PTK)activitydimerize(homo and heterodimers)auto-,trans-phosphorylationrecruit signaling molecules at specified phosphorylated sitesAberrantly expressed in many tumorsGFGFPPPPPGFPPPPReceptor Protein Tyrosine Kinase Signaling ACollection of Receptor protein Tyros
23、ine Kinases(RPTK/RTK)GROWTH FACTORRECEPTORTYROSINEKINASEGRB-2SOSRASRAFMAPKKMEMBRANEMAPKDimerization;autophosphorylationInteraction ofsignaling moleculesActivation of“downstream”kinasesJAKSTATPNUCLEUSMAPKPhosphorylation of transcription factorsSTATGENE EXPRESSION,i.e.,fos,jun,mycCYTOPLASMPPPPPPPPPPPP
24、PPPP PPPPPRPTK SIGNALING CASCADES25PTK(Proto-oncogene)Viral oncogene*(viral oncoprotein)Oncogenic alterationTumour/cancer types(only the most frequent,Mainly human types are described)EGFR/ErbB1(c-erbB)V-erbB fromAEV(p68/74abB)V-erbB:Truncated EGFR PTKC-erb:Overexpression(amplification)Extracellular
25、 domain deletionsv-ErbB:fibrosarcomasc-ErbB:mammary carcinoma,glioblastoma multiforme,Cvarian,non-small-cell lung and lther cancersErbB2/HER2/NeuOverexpression(amplification)No recurrent human mutations(Val664glu in rodents)Mammary,ovarian,gastric,non-small-cell lung and colon cancerErbB3/HER3Overex
26、pression;constitutive tyrosine phosphoylation(heterodimer with ErbB2)Mammary carcinomaErbB4/HER4Overexpression Mammary carcinoma,granulasa cell tumoursIGF-1ROverexpression(expression required for in vitro transformation by many oncogenes and DNA viruses)Cervical and other carcinomas,sarcomasPDGFR-Ov
27、erexpression(amplification)Glioma,glioblastoma,ovarian carcinomaPDGFR-Tel-PDGR-(t(5;12)translocation fusing Ets-like Tel with PDGFR-PTK domain)OverexpressionTel-PDGFR-:chronic myelomonocytic leukaemiaPDGFR-:gliomaCSF-1R(c-fms)V-fms fromFesV(p170gag-fms)v-fms:Truncated CSF-1R PTK with mutant C-termin
28、al tailConstitutively activec-fms:GOF point mutationsOverexpression v-fms:jeline sarcomasc-fims:acute and chronic myelomonocytic leukaemias,monocytic tumours,malignant histiocytosis,endometrial cancer,gliomaKit/SCFR(c-kit)V-kit fromFeSV(p80gag-kit)v-kit:Truncated Kit/SCFR PTK with mutant C-terminal
29、tailConstitutively activec-kit:GOF point mutations and small deletionsOverexpressionV-kit:feline fibrosarcomasC-kit:malignant gastrointestinal stromal tumours,acute myeloid leukaemias,myelodysplastic syndromes,mast-cell leukaemia/systemic mastocytosis,seminomas/dysgerminomas,small-cell lung cancer a
30、nd other carcinomasReceptor Protein Tyrosine Kinases and CanceractinCIP4DNAFABDFERMkinaseKinase-likePHSH2SH3Actin-binding domainBtk motifCdc42-bindingCIP4 homology domainDNA-binging domainFocal adhosion-binding domainIntogcyin-binding domainPIK domainPscodo PIK domainPlockstrin homology domainSrc ho
31、mology-2 domainSic homology-3 domainSH2kinaseSH3DNAactinSH2kinaseSH3FERMKinase-likekinasekinaseSH3SH3SH2kinasekinaseFERMFABDkinaseCIP4SH2SH3kinaseSH2SH3SH2kinaseSH2SH2kinasePHSRCABLJAKACKCSKFAKFESFRKTECSYKFGR,FYN,SRC,YES1,BLK,HCK,LCK,LYNBAL,ARGJAK1,JAK2,JAK3,TYK2ACK1,ACK2CSK,MATK/CTKFAK,FESFA,PYK2BR
32、K,FRK,SRMSBMX,BTK,ITK,TEC,TXXSYK,ZAP70Non-receptor Protein Tyrosine Kinase Signaling BPTKPTK(Proto-oncogene)(Proto-oncogene)Viral oncogene*Viral oncogene*(viral oncoprotein)(viral oncoprotein)Oncogenic alterationOncogenic alterationTumour/cancer types(only the most frequent,Tumour/cancer types(only
33、the most frequent,Mainly human types are described)Mainly human types are described)ROS(c-ros)v-ros fromavianUR2 SV(p68gag-ros)v-ros:Truncated Ros PTK domain.Constitutively activec-ros:OverexpressionRare truncations/point mutations?v-ros:avian fibrosarcomasC-ros:glioblastomas,astrocytomasAlkNPM-Alk(
34、t(2;5)nucleophosmin fused to Alk PTK domain)lg-Alk(t(2;22)lg fused to Alk PTK domain)Other sporadic fusions with tropomyosin,etc.Non-Hodgkin lymphomas,CD30+and CD30-anaplastic large-cell lymphomaSrc(c-src)v-src fromRSV(pp60v-src)v-src:C-teminal truncation and point mutations(increased kinase activit
35、y)c-src:C-terminal truncation(increased kinase activity)Overexpression and/or increased kinase activitypp60v-sre:avian sarcomasc-Src truncation:colon cancerc-Src overexpression:mammary and pancreatic cancers.neuroblastomas,othersAbl(c-abl)V-abl fromAbelson MLV(p160gag-abl)or fromP1-FeSVv-abl:N-termi
36、nal(SH3)truncation ot 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:3 breakpoint cluster regions in BCR.The chimaeric mRNA usually starts with exon a2 of ABL,it never includes exon 1a or 1b.Tel-Abl(t(12;22)N-terminal(H-L-Hregion of Tel fused
37、 with Abl exon 2ap160gag-abl:murine acute leukaemiasp190Bct-Abl:50%of Ph+acute lymphocytic leukaemias,rarely chronic myelomonocytic leukaemiasp210Bcr-Abl:chronic myeloid leukaemias,30%of Ph+acute lymphocytic leukaemiasp230Bcr-Abl:some Ph+chronic neutrophilic leukaemiasTel-Abl:rare cases of acute lym
38、phocytic leukaemiasNon-receptor Protein Tyrosine Kinases and CancerSrc StructureBasal Activty(discerned from crystal structure)Activated KinaseSrc domains and Src activityPI-3-kinaseSrcSrcFamilyFamilyrasmycmitosismitoticfunctionsstresspathwaysextracellularmatrixcytoskeletalreorganizationantigensoxid
39、ativestresscytokinesGproteincoupledreceptorsRPTKsangiogenesisThe World According to SrcRAS superfamlyCGTPase proteins1.H-ras,chr11;K-ras,chr12;N-ras,chr12.5 exons,188-189 aa,MW 21 KD3.GTPaseRAS signaling RASPI3KPIP3Akt/PKB Rho-GEFRaf(MAPKKK)MEK(MAPKK)Erk1/2(MAPK)Mnk1,RSK,Ets,Elk-1,SAP-1Ral-GEFRal-AR
40、al-BCdc42,RacThe World According to RASRAS mutation and CancerCatalyticregPHT308*S473*Lipid bindingSer/thr kinase*Full activation of kinase requires phosphorylation of both T308 and S473Akt structureIntracellular Serine/Threonine Kinase SignalingDCatalyticregPHT308S473PI3KactivationPI(3,4,5,)P3PDK1P
41、PPDK2,PDK1,ILKCatalyticregPHT308S473PPPI(3,4,5,)P3NUCLEUSGrowth factor receptorsPI(4,5,)P2Akt activationCatalyticregPHT308S473GSK3PFK-2PDE-3BmTORIkBBadp21ForkheadGlycogensynthesisProteinsynthglycolysiscAMPtranslationExpression of Fas ligandExpressionOf antiapoptotic genesBcl-XLBcl-2Cell cycleAkt/PKB
42、-mediated signalsPI3K and MEK pathway in cancer1.c-myc,8q24,439aa;N-myc,2p23-24,456aa;L-myc,1p32,364aa2.Nuclear transcription factorNuclear transcription factorEMycMYC regulation二、抑癌基因二、抑癌基因 Tumor Suppressor GeneEvidences for Tumor Suppressor Genes Somatic Cell Genetic Studies Somatic Cell Genetic S
43、tudies 1969,Ephrussi and HarrisRas oncogene-NIH3T3-transformationRas oncogene-CHO-NO transformation DNA transfection DNA transfection1.抑癌基因的发现抑癌基因的发现PEDIGREE of a family with familial retinoblastoma was published by Thaddeus P.Dryja and his collaborators.Affected members are indicated by solid circl
44、es(females)or squares(males).Five children in the second generation developed the tumor.One son who was unaffected had nonetheless inherited a mutated chromosome 13:two of his daughters were affected.13q14 Cytogenetic Analysis of familial cancers Cytogenetic Analysis of familial cancersCloning and I
45、dentification of RB gene1986 Cloning of Rb genePut Rb gene back into Rb cells-lose transforming ability such as tumorigenicity,soft agar colony forming ability1st tumor suppressor gene identified!Terminology:Tumor suppressor genes,Anti-oncogenes,Recessive oncogenesTumor Suppressor Gene：a gene whose
46、product can negatively control cell growth and suppress cell transformation.1.Negatively control cell growth and division1.Negatively control cell growth and division1.Negatively control cell growth and division2.Suppress cell transformation2.Suppress cell transformation2.Suppress cell transformatio
47、n3.Mutated and inactivated in cancer3.Mutated and inactivated in cancer3.Mutated and inactivated in cancer4.tissue-specific 4.tissue-specific 4.tissue-specific Criteria:Criteria:A.Expresses in normal tissuesA.Expresses in normal tissuesA.Expresses in normal tissuesB.Mutated in cancerB.Mutated in can
48、cerB.Mutated in cancer：DNA,mRNA,proteinDNA,mRNA,proteinDNA,mRNA,proteinC.Re-introduction make cancer cell lose transforming abilityC.Re-introduction make cancer cell lose transforming abilityC.Re-introduction make cancer cell lose transforming abilityFeatures:Features:2.抑癌基因定义抑癌基因定义Knudsons Two-hit
49、theoryDefinition:inactivation of one allele by mutations or small deletions and loss of the second allele,usually by chromosome loss(loss of heterozygosity).-chromosome loss -mutations -small intragenic deletions -promoter methylation(epigenetics)Haploinsufficiecy Inactivation of one allele is enoug
50、h to lead to tumor growth3.抑癌基因失活机制抑癌基因失活机制Epigentic deregulation The methylation states of promoter and histone affect the expression level of gene 11stst hit hit22ndnd hit hitmutMemutmutMeMeMeMeMutationMethylationLoss MethylationLoss MethylationMutation Mutation Methylation Methylation+LOH methyla