癌生物学-赵莹珺（癌基因与抑癌基因）

1、赵莹珺复旦大学生物医学研究院复旦大学附属肿瘤医院/肿瘤研究所 2015年度癌生物学课程提提 纲纲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 growth1.1.癌基因的发现癌基因的发现Non-Transforming (Avian Leukemia Viruses, ALV)1, Induce leukemia after long latency periods2, Do not “transform” tissue cu

2、lture cellsVirus and CancerTransforming (Sarcoma Viruses, RSV)-cell free lysates could induce sarcomas in other chickens1，Acute; 2，Transform cultured cells病毒致癌病毒致癌- Nobel Prize! 1966BRSV (gag, pol, env, src)R.T.(1975 Nobel)gag, pol envsrccDNARSV (td: gag, pol, env)gag, pol, envDenature and Hybridize

3、1975,1977unhybridizedsequenceshybridizedsequencesgag, pol, 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 oncogene1980, 1st oncogene identifiedSrc （v-onc, v

4、iral oncogene）Human Bladder Tumor cell line DNAIsolate high MW DNAIsolate DNA fragmentsRestriction endonucleaseTransfectionNIH 3T3fibroblastsTransformationIsolate DNA (99% mouse +8-10 human genes)TransformationIdentify Human DNAActivated Proto-oncogenes from DNA transfectionDRESULT: RAS “Activation”

5、is due to a SINGLE point mutation (gly val) at codon 12Use Alu 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, Rober

6、t Weinberg, etc three groups2.癌基因定义癌基因定义 Definitiona 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

7、 into the viral genome. 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. a normal cellular form of a gene that controls cell prolife

8、ration 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1Controls cell proliferation and survival;2Can be converted into oncogene,

9、and induce transformation3Conserved across organisms4Tissue-specificPhysiological function: cell signaling pathways tightly controlled“Hyper”-functional3.原癌基因激活机制原癌基因激活机制Transduction via retrovirusesLTRLTRViral RNAPackagingOf retrovirusgag pol envProto-oncogene is “captured” or “usurped” from host c

10、ell genomeRetrovirus without oncogeneLTRLTRgag pol env SrcRetrovirus with oncogeneRetroviral promoter/enhancer insertion Chronic Myelogenous Leukemia (CML)：chromosome 9q34 (c-abl) chromosom22q11.2 (bcr)，proto-oncogene c-abl is activatedChromosomal translocationq349535322q11.2ablder 9Phabl9q3422q11.2

11、22q11.29q34Der (22)DNA RNA Proteinbcrbcrbcr/abl Fusion protein Gain of multiple copies of defined chromosomal regions (1) Homogeneously staining region (HSR) (2) Double minute chromosomes (DM) Tumor OncogeneAmplificationSmall cell lung cancer c-myc Up to 8OX N-myc Up to 50X L-myc Up to 20XNeuroblast

12、omas N-myc Up to 250XGlioblastomas c-erbB1 (EGFR) Up to 50XMammary carcinoma c-erbB2 (HER2) Up to 30X c-myc Up to 50X Cyclin D1 Up to 30X AmplificationCellular Proto-oncogenes amplified in human tumorsHSRDMRas：P21 transforming protein c-H-ras-Harvey rat sarcoma virus c-K-ras-Kirsten rat sarcoma viru

13、s N-ras-NeuroblastomaPoint mutationHot mutation points: 12, 13, 61 12 Gly-Valwt-RasGTPwt-RasGDPActive formInactive formmt-RasGTPmt-RasGDPConstitutively activegrowth factors(sis)GTPase proteins(ras)guanine nucleotideExchange proteins (rho)Cytoplasmic Membrane associated Tyrosine kinases (src)growth f

14、actor 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 mobilisatio

15、n 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,

16、fms)C. Intracellular 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 genes a- Protein Tyrosine Kinase (src, yes, fps, abl, met )b- Protein -Serine/Threonine kinases (akt, mos, raf)c- R

17、as proteins (Ha-ras, Ki-ras, N-ras)d- 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 II a- jun, fos b- my

18、c, N-myc, myb, ski, relBasic Cellular Signaling Machinery: for example Kinase signaling and CancerReceptor Protein Tyrosine Kinase Signaling Non-receptor Protein Tyrosine Kinase Signaling Intracellular Serine/Threonine Kinase Signaling5.癌基因功能癌基因功能ABCGTPase proteinsDNuclear transcription factorE tran

19、s-membrane (glyco)proteins possess intrinsic protein tyrosine kinase (PTK) activity dimerize (homo and heterodimers) auto-, trans-phosphorylation recruit signaling molecules at specified phosphorylated sites Aberrantly expressed in many tumorsGFGFPPPPPGFPPPPReceptor Protein Tyrosine Kinase Signaling

20、 ACollection of Receptor protein Tyrosine Kinases (RPTK/RTK)GROWTH FACTORRECEPTORTYROSINEKINASEGRB-2SOSRASRAFMAPKKMEMBRANEMAPKDimerization;autophosphorylationInteraction ofsignaling moleculesActivation of “downstream”kinasesJAKSTATPNUCLEUSMAPKPhosphorylation of transcription factorsSTATGENE EXPRESSI

21、ON, i.e., fos, jun, mycCYTOPLASMPPPPPPPPPPPPPPPP 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-

22、erb:Overexpression (amplification)Extracellular 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, gastr

23、ic, non-small-cell lung and colon cancerErbB3/HER3Overexpression; 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

24、 DNA viruses)Cervical and other carcinomas, sarcomasPDGFR- Overexpression (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-

25、fms fromFesV (p170gag-fms)v-fms:Truncated CSF-1R PTK with mutant C-terminal 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

26、)V-kit fromFeSV(p80gag-kit)v-kit:Truncated Kit/SCFR PTK with mutant C-terminal tailConstitutively activec-kit:GOF point mutations and small deletionsOverexpressionV-kit: feline fibrosarcomasC-kit: malignant gastrointestinal stromal tumours, acute myeloid leukaemias, myelodysplastic syndromes, mast-c

27、ell leukaemia/systemic mastocytosis, seminomas/dysgerminomas, small-cell lung cancer and other carcinomasReceptor Protein Tyrosine Kinases and CanceractinCIP4DNAFABDFERMkinaseKinase-likePHSH2SH3Actin-binding domainBtk motifCdc42-bindingCIP4 homology domainDNA-binging domainFocal adhosion-binding dom

28、ainIntogcyin-binding domainPIK domainPscodo PIK domainPlockstrin homology domainSrc homology-2 domainSic homology-3 domainSH2kinaseSH3DNAactinSH2kinaseSH3FERMKinase-likekinasekinaseSH3SH3SH2kinasekinaseFERMFABDkinaseCIP4SH2SH3kinaseSH2SH3SH2kinaseSH2SH2kinasePHSRCABLJAKACKCSKFAKFESFRKTECSYKFGR,FYN,S

29、RC,YES1,BLK,HCK,LCK,LYNBAL,ARGJAK1, JAK2, JAK3, TYK2ACK1, ACK2CSK, MATK/CTKFAK, FESFA, PYK2BRK,FRK, SRMSBMX,BTK,ITK,TEC,TXXSYK, ZAP70Non-receptor Protein Tyrosine Kinase Signaling BROS(c-ros)v-ros fromavianUR2 SV(p68gag-ros)v-ros:Truncated Ros PTK domain. Constitutively activec-ros:OverexpressionRar

30、e truncations/point mutations?v-ros: avian fibrosarcomasC-ros: glioblastomas, astrocytomasAlkNPM-Alk (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 ly

31、mphomaSrc(c-src)v-src fromRSV(pp60v-src)v-src:C-teminal truncation and point mutations (increased kinase activity)c-src:C-terminal truncation (increased kinase activity)Overexpression and/or increased kinase activitypp60v-sre:avian sarcomasc-Src truncation: colon cancerc-Src overexpression: mammary

32、and pancreatic cancers.neuroblastomas, othersAbl(c-abl)V-abl fromAbelson MLV(p160gag-abl)or fromP1-FeSVv-abl:N-terminal ( 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 ch

33、imaeric 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 with Abl exon 2ap160gag-abl: murine acute leukaemiasp190Bct-Abl: 50% of Ph+ acute lymphocytic leukaemias, rarely chronic myelomonocytic leukaemiasp210Bcr-Abl: chron

34、ic myeloid leukaemias, 30% of Ph+ acute lymphocytic leukaemiasp230Bcr-Abl: some Ph+ chronic neutrophilic leukaemiasTel-Abl: rare cases of acute lymphocytic leukaemiasNon-receptor Protein Tyrosine Kinases and CancerSrc StructureBasal Activty (discerned from crystal structure)Activated KinaseSrc domai

35、ns and Src activityPI-3-kinaserasmycmitosismitotic functionsstress pathwaysextracellular matrixcytoskeletal reorganizationantigensoxidative stresscytokinesG protein coupled receptorsRPTKsangiogenesisRAS superfamlyCGTPase proteins1. H-ras, chr11; K-ras, chr12; N-ras, chr12. 5 exons, 188-189 aa, MW 21

36、 KD3. GTPaseRAS signaling RASPI3KPIP3Akt/PKB Rho-GEFRaf (MAPKKK)MEK(MAPKK)Erk1/2(MAPK)Mnk1,RSK,Ets,Elk-1, SAP-1Ral-GEFRal-ARal-BCdc42, RacRAS mutation and CancerCatalyticregPHT308*S473*Lipid bindingSer/thr kinase*Full activation of kinase requires phosphorylation of both T308 and S473Akt structureIn

37、tracellular Serine/Threonine Kinase SignalingDCatalyticregPHT308S473PI3KactivationPI(3,4,5,)P3PDK1PPPDK2, PDK1, ILKCatalyticregPHT308S473PPPI(3,4,5,)P3NUCLEUSGrowth factor receptorsPI(4,5,)P2Akt activationCatalyticregPHT308S473GSK3PFK-2PDE-3BmTORIkBBadp21ForkheadGlycogensynthesisProteinsynthglycolys

38、iscAMPtranslationExpression of Fas ligandExpressionOf antiapoptotic genesBcl-XLBcl-2Cell cycleAkt/PKB-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 regulationEvidences f

39、or Tumor Suppressor Genes 1969, Ephrussi and HarrisRas oncogene-NIH3T3-transformationRas oncogene-CHO-NO transformation1.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 circles (females)

40、 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. 13q14Cloning and Identification of RB gene1986 Cloning of Rb genePut Rb gene back into Rb cells - lose tr

41、ansforming 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 product can negatively control cell growth and suppress cell transformation. 2.抑

42、癌基因定义抑癌基因定义Knudsons Two-hit 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)Haploinsufficiec

43、y Inactivation of one allele is enough 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 1st hit2nd hitmutMemutmutMeMeMeMeMutationMethylationLoss MethylationLoss MethylationMutation Mutation Methylation

44、Methylation + + + + LOH methylation Chromosome loss MethylationCancer syndromeGenePrincipal TumorsProtein Product LocalizationMode of ActionRetinoblastomaRB1Retinoblastoma. OsteosarcomaNucleusTranscriptonal regulator/ factorLi-Fraumenip53Sarcomas. breast and brain tumorsNucleusTranscripton factorFam

45、ilial adenomatous polyposisAPCAdenomatous polyps. colon cancerCytoplasmRegulates -catenin functionWilms tumorWT-1NephroblastomaNucleusTranseription factorNeuofibromatosis type 1NF-1Neurofibromas. sarcomas. GliomasCytoplasmP21 ras-GTPase activatorFamilial melanoma and pancreatic cancerp16Melanoma, pa

46、ncreatic cancer.NucleusCyclin-dependent kinase inhibitorFamilial breast cancer BRCA1BRCA2Breast and ovarian cancer Breast NucleusUnknownDNA repair, chromosomal stabilityTumor Suppressor Genes Associated With a Cancer SyndromeReceptor：PTCH DCCIntermediate regulators：NF1 PTEN APC NF2Transcription fact

47、ors/activators：p53 WT1 RB1 VHLCell cycle inhibitors：p16 p21 p15DNA repair：MSH2 MLH1 PMS2 ATM BRCA1 BRCA2According to localization and molecular function4.抑癌基因分类抑癌基因分类Gatekeepers vs. CaretakersPTEN, p53, RB5.抑癌基因功能抑癌基因功能1. 17p13.12. 11 exons, 393 aa, MW 53 KD3. Transactivator (TA)Transcriptional Acti

48、vation domainDNA binding domainTetramerization domainGene and protein structureTP53Mutations in cancers: Loss of Heterozygosity Colorectal cancers Inhibition of transformationA p53 null mouse viable loss of one or both p53 alleles gives a tumor phenotype loss of p53 cooperates with other alterations

49、 to increase tumor incidencep53: Li-Fraumeni Syndrome- an inherited predisposition to cancer - various kinds of cancers occur: osteosarcomas, soft tissue sarcomas, etc- multiple tumors in the same individual A tumor suppressor geneClassification of p53 mutationsMutant: loss of functionWide type: los

50、s of functionp53p53p53p53Transcriptional activationMutantp53p53p53p53Binds and regulates transcriptionof other genes MDR-1, c-mycp53 functionsTranscriptional Activator: binds to cis- element Cell cycle regulatory genes p21/WAF1/Cip1, GADD45, cyclin GApoptosis genes Bax, Puma, Noxa, etcRegulators of

51、itself Mdm2, ARFInactivation of p53Ubiquitination and degradation by Mdm2 (E3 ligase)p53mdm2p53E2E1p53UBUBmdm2Interaction with virus proteins:SV40 large T Ag, HPV16E6, E1BT AgDNA bindingDominant-negativeMutant p53Activation of p53: increase of stabilityInhibition of Mdm2p53Mdm2p19ArfPhosphorylation

52、of p53 Ultraviolet light Ionizing radiation Drugs (cisplatin, adriamycin) ATMATRChk1Chk2DNA-PKp53pppppmdm2outAcetylation of p53 Acetyltransferase: p300/CBP, PCAF, etc Sumoylation of p53(Small ubiquitin related molecules)p53asThe p53 signaling networkPTEN ( phosphatase and tensin homolog deleted on c

53、hromosome 10, PTEN); MMAC1 (mutated in multiple advanced cancers 1);TEP1 (TGF-regulated and epithelial cell-enriched phosphatase 1)Gene structurePTEN/MMAC1, discovered in 1997Regulation of PTENA. Constitutive Phosphorylation (by casein kinase 2)C. Ubiquitination (proteasome degradation)B. Lipid inte

54、raction (PIP2)(Membrane localization) D. Protein interactions (stability, etc)PI 3.4.5 P3 PI - 4.5 P2PTENPI3KPTEN activityCanonical PTENPI3KAKTmTOR pathway Biological functions of PTEN in tumorigenesisPTENAktForkheadp27 Down-regulationGSK-3cyclin D1 degradation2. Cell cycle arrest in G1 phaseRac, Rh

55、oMMP-23. Inhibits migration and invasionmigrationinvasionHIF-1VEGFangiogenesis4. Inhibits angiogenesismTORelF4E disfunction1. protein synthesisinhibitionPTEN in cancerSchematic of RB in cell cycle control.Alterations of the CdKRBE2F pathway in human cancer三、癌基因三、癌基因/抑癌基因与多步骤抑癌基因与多步骤癌变癌变Synergy of On

56、cogenes & Tumor Suppressor Genes in Multi-step Tumorigenesis 1. Multi-hit requirements for carcinogenesisNIH3T3 transfected assay is an one-hit mechanism (Ras transformation)Epidemiology supports multi-hit requirements for most tumorsOnly 20% of human tumor DNA produce foci in NIH3T3 transfectio

57、n assayProblems:NIH3T3 is a immortal cell line. NIH3T3 cell is not exactly normal !How about foci-forming assays using embryo fibroblasts ?REFNO FOCI !Ras GeneREFNO FOCI !v-myc Gene+ RasFOCICONCLUSION: At least two genes are required for transformation of “normal” cells ESTABLISHMENT(IMMORTALIZATION

58、) GENESc-myc, N-mycE1A (adenovirus)Large T (polyoma)NUCLEARTRANSFORMING GENESc-K-ras, C-H-rasN-rasc-neuCYTOPLASMICTumor formation can be mimicked in the lab. By delivery of the following genes:1.Catalytic hTERT subunit of telomerase: Maintains telomere length2.SV40 large T antigen: inactivates both

59、the p53 and Rb tumor suppressor gene3.Activated oncogenic ras oncogene: induces transformation to a cancerous state allowing cells to grow indefinitely in the absence of growth factorsDisruption of the intracelluar pathways regulated by the large-T, oncogenic ras and telomerase suffice to create a h

60、uman tumor cell.Hahn, W.C., Counter, C.M., Lundberg, A.S., Beijersbergen, R.L., Brooks, M.W., and Weinberg, R.A.Nature 400, 464-468, 19991. Whitehead Institute for Biomedical Research2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142Cancer Cell, Vol. 6, 171-183,