6、肿瘤分子生物学

肿瘤分子生物学郑国沛副研究员Email:zhengguopei@126.com广州医科大学附属肿瘤医院/肿瘤研究所2017-01肿瘤分子生物学-细胞信号转导—生命的表述语言细胞信号转导研究是当前生命科学领域的一个核心细胞信号转导是研究细胞感受并转导环境(也包含内环境)刺激，从而调节代谢生理反应和基因表达的分子途径的一门科学。生命科学研究论文发表最集中的领域之一全球药物开发四分之一集中的领域应用分子生物学理论阐明肿瘤的发生、发展及其本质，运用分子生物学技术研究肿瘤相关基因及其表达产物在肿瘤发生发展中的作用，为肿瘤的预防、诊断和治疗提供新措施，从根本上提示肿瘤的发生发展机制。肿瘤分子生物学HistoryOverview1951 NGF(1stgrowthfactor)1957 cAMP(1971Nobel)Phosphorylation;Proteinkinase(1992Nobel)

1960 EGF(1980Nobel)1970’ Gprotein;Gprotein-coupledreceptor(1994Nobel)1978 Src(1stproteintyrosinekinase)1986 Rb(1sttumorsuppressorgene)1990’ Cross-talkNobelPrizesinSignalingStudy信号转导与肿瘤一、信号转导概念和组成特征二、肿瘤信号转导的异常三、肿瘤干细胞和EMT四、以信号转导为靶的肿瘤治疗五、常用的信号转导研究方法六、肿瘤信号转导研究实例细胞信号转导指细胞通过细胞表面（或胞内）受体接受外界信号，通过系统级联传递机制，将胞外信号转导为胞内信号，最终引起细胞生理反应或特定基因的表达，引起细胞伯应答反应，这种特定的反应系统称之为细胞信号通路一、信号转导的概念和组成特征概念信号受体

信号转导级联通路细胞核（转录因子染色体）基因表达蛋白质表达谱的改变信号传导过程生物效应信号识别信号转导ExtracellularfactorsMembranereceptorsAdaptorproteinsSecondmessengersIntracellularkinasesTranscriptionfactors基本组成刺激细胞增殖的因子1) GrowthFactors

(家族性；受体为酪氨酸激酶；相对特异性；多样性）2) Cytokines(ILs,CSFs,IFN)

(受体不具酪氨酸激酶活性） 3)Hormones,Neurotransmitters (通过GPCR传递信号）其它细胞外因子 TNFs,Adhesionmolecules(Fibronectin,Laminin, Collagen)ExtracellularFactors生长因子受体

酪氨酸激酶(tyrosinekinase)

EGFR,IGFR,PDGFR,FGFR,NGFR,HGFR

G蛋白耦联受体(GPCR)

最大的受体家族 7-transmembrane

无激酶活性，通过G蛋白传导信号细胞因子受体 1）淋巴细胞表面受体(TCR,BCR) 2)白介素受体 3）细胞凋亡受体(Fas,TNFR)粘附因子受体

1）Cadherins 2)Integrins 3)Igsuperfamily 4)SelectinsMembraneReceptors生长因子受体142012EGFRsignaling

本身不具有催化活性，在信号转导通路中起着重要的桥梁作用，将被配体激活的受体与其下游的信号转导分子相连接，沟通整条信号转导通路。AdaptorProteinsAdaptorProteinDomainsPTB:Phosphotyrosinebindingdomain(BindP-tyrosine)SH2:Src-homologydomain2(BindP-tyrosine)SH3:Src-homologydomain3(BindProline-richmotif(PXXP))PH:Pleckstrinhomology(PIP2phospholipidbinding)WW:BindPXPX,P-serineorP-threoninePDZ:Bindhydrophobic residuesatC-terminusFYVE:BindPtdIns-3P

DD:Deathdomain;apoptosis 定义：细胞外因子与受体结合后，在细胞内产生的具有生物活性的小分子。

种类：cAMP,cGMP,DAG,IP3,Ca2+

功能：

cAMPPKA cGMPPKG DAGPKC Ca2+(calmodulin)CAMKI,II,III&MLCKSecondMessengersVeryimportantforsignalingSerine/ThreoninekinasesImportantkinases

Mitogen-activatedproteinkinse(MAPK) ProteinKinaseA(activatedbycAMP) ProteinKinaseG(activatedbycGMP) ProteinKinaseC(activatedbyDAG) CAMK(activatedbyCa2+/Calmodulin) PI3K(Growthfactorreceptor)IntracellularKinases特异性网络性，多样性通路之间的多层次交流(cross-talk)不同的信号可以激活同一条转导通路同一信号往往能够激活多条转导通路二、信号转导系统的特征MAPKPathwayBiologicalEffectProliferationStressApoptosisInflammationRAS-RAF-MEK-MAPK•Ligandbindsreceptor•LigandbindsreceptorPTK•AutophosphorylationontyrosinePPPPRAS-RAF-MEK-MAPK•LigandbindsreceptorPTK•Autophosphorylationontyrosine•GRB2(aSH2-andSH3-containingprotein)bindstothereceptorphosphotyrosinemotifY-V/L-N-XviaitsSH2domainPPPPRAS-RAF-MEK-MAPKSH2SH3GRB2SOS•LigandbindsreceptorPTK•Autophosphorylationontyrosine•GRB2(aSH2-andSH3-containingprotein)bindstothereceptorphosphotyrosinemotifY-V/L-N-XviaitsSH2domain•TheSH3ofGRB2bindsconstitutivelytotheproline-richsequenceintheC-terminusofSOS(aguaninenucleotideexchangefactorforRAS).

PPPPSH2SH3GRB2SOSRAS-RAF-MEK-MAPK•RecruitmentofSOStothecloseproximityofRASinthemembranePPPPSH2SH3GRB2SOSRASGDPRAS-RAF-MEK-MAPK•RASbecomesactivatedbyexchangingGDPforGTPPPPPSH2SH3GRB2SOSGDPGTPRASRAS-RAF-MEK-MAPK•TheRAS-GTPeffectordomaininteractswiththeN-terminalregulatoryregionoftheRAF(serine/threonineproteinkinase),hencerecruitingRAFtothemembranePPPPSH2SH3GRB2SOSRASGTPRAFRAS-RAF-MEK-MAPK14-3-3•ActivationofRAF(mostlikelybyphosphorylationofRAFandbindingtothescaffoldprotein14-3-3)PPPPSH2SH3GRB2SOSRASGTPRAFRAS-RAF-MEK-MAPK14-3-3•ActivatedRAFinturnactivatesMEK(alsocalledMAPKkinase;adualspecificitykinase)byphosphorylationontwoconservedserineresiduesinMEK.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPRAS-RAF-MEK-MAPK14-3-3•ActivatedMEKactivatesMAPK(aserine/threonineproteinkinase)byphosphorylationofconservedthreonineandtyrosineresidues.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPRAS-RAF-MEK-MAPK14-3-3•ActivatedMAPKphosphorylatesanumberofsubstratesintheplasmamembraneandthecytoplasm;PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPMAPKPPSubstratesSubstratesPPRAS-RAF-MEK-MAPK14-3-3•ActivatedMAPKphosphorylatesanumberofsubstratesintheplasmamembraneandthecytoplasm;italsotranslocatedintothenucleus(withinmin)whereitphosphorylatesnucleartranscriptionfactors.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPSubstratesRAS-RAF-MEK-MAPKMAPKPPMAPKPP14-3-3•ActivatedMAPKphosphorylatesanumberofsubstratesintheplasmamembraneandthecytoplasm;italsotranslocatedintothenucleus(withinminutes)whereitphosphorylatesnucleartranscriptionfactors.Transcriptionofgenesimportantforcellproliferation.PPPPSH2SH3GRB2SOSRASGTPRAFMEKPPRAS-RAF-MEK-MAPKSubstratesMAPKPPMAPKPPPDephosphorylationbytyrosinephosphatasesBindingofinhibitoryproteinsEndocytosisDegradationTerminationofSignalTransductionPKC:ProteinkinaseCPTP:ProteintyrosinephosphataseUb:Ubiquitin二、肿瘤信号转导的异常多阶段(Multiplestages)：激发、促进、进展和转移多通路(Multiplepaths)：增殖、分化、凋亡、侵袭与转移多基因(Multiplegenes)：瘤基因、抑瘤基因、损伤修复相关基因、细胞周期调控基因肿瘤的发生是一个涉及多个阶段、多条通路、多个基因的病理过程肿瘤的本质特征自己自足的生长信号抗生长信号不敏感抵抗细胞死亡潜力无限的复制能力持续的血管生成组织浸润和转移避免免疫摧毁促肿瘤炎症细胞能量异常基因组不稳定和突变Self-SufficiencyinGrowthSignalsInsensitivitytoAntigrowthSignalsResistingCellDeathLimitlessReplicativePotentialSustainedAngiogenesisTissueInvasionandMetastasisAvoidingImmuneDestructionTumorPromotionInflammationDeregulatingCellularEnergetics1.RoleofTGF-binCarcinogenesis2.NF-kBinCarcinogenesis4.ErbB/HERinCarcinogenesis5.PI3K/AktinCarcinogenesis三、肿瘤干细胞和EMT肿瘤研究新困惑当前肿瘤生物学三大困惑:众多肿瘤中存在传统克隆演进学说或微环境差异所不能解释的表型和功能异质性；以肿瘤来源建立稳定细胞系的不容易性；现细胞细胞行再成瘤时所需细胞数量巨大性。肿瘤干细胞有助于阐释上述问题-Rare-Normallylocalizedina‘protected’environmentcalledNiche,wheretheyonlyoccasionallydivide.-ButtheypossessHighproliferativepotentialandcangiverisetolargeclonesofprogenyinvitroorinvivofollowinginjuryorappropriatestimulation.-PossesstheabilitytoSelf-Renew(i.e.,asymmetricorsymmetriccelldivision)-Cangenerate(i.e.,Differentiateinto)oneormultipleorallcelltypes(uni-,oligo-,multi-,pluri-,ortoti-potent).CharactersofStemCellsCommittedprogenitorcellssymmetricSCrenewalasymmetriccelldivision(ACD)symmetricSCcommitment(differentiation)SCTang,CellRes.2012PrimitiveectodermTrophectodermPrimitiveEndodermHowcanhEScellsbederived?EScellsHSCscanbesubdividedintolong-termself-renewingHSCs,short-termself-renewingHSCsandmultipotentprogenitors(redarrowsindicateself-renewal).Theygiverisetocommonlymphoidprogenitors(CLPs;theprecursorsofalllymphoidcells)andcommonmyeloidprogenitors(CMPs;theprecursorsofallmyeloidcells).BothCMPs/GMPs(granulocytemacrophageprecursors)andCLPscangiverisetoallknownmousedendriticcells.ErP,erythrocyteprecursor;MEP,megakaryocyteerythrocyteprecursor;MkP,megakaryocyteprecursor;NK,naturalkiller.DevelopmentofHematopoieticStemCellsStemCellsMultipotentProgenitorsOligolineageProgenitorsMatureCellsReyaetal.2001Nature414:105-111Tang,CellRes.2012Wheredidtumorcellscomefrom?Fromthestandpointoftransformationprobability,theST-SCthatretainsself-renewalactivityandprogenitorcellsthatarehighlyproliferative(demarcatedbytwoverticalthicklines)theoreticallycouldrepresentthebesttargetsfortumorigenictransformation

CSCsdefinitionsandterminologyCSCs,(Tumorstemcells,TSCs;tumorinitiativecells,TICs)arerarecellsincancersexhibitingstemcellscharacteristics,particularlytheabilitytoself-renewalandtogiverisetoahierarchyofprogenitoranddifferentiatedcells;

TheconceptofCSChasbeendemonstratedinseveralhumancancersincludingleukemia,braintumor,breastcancer,prostatecancer,lungcancer,pancreascancer,coloncancer,andsoonMilestonesinaconceptofcancerstemcells1855

VirchowRproposedthe‘‘embryonal-resthypothesis’’of

tumorformation,basedonhistologicalsimilaritiesbetween

tumorsandembryonictissues.Thistheory,laterexpandedby

otherpathologistssuchasJuliusConheim,whosuggestedthat

tumorsdevelopfromresidualembryonictissues(胚胎残余假说)1994

LapidotTandcolleaguesshowacellinitiatinghumanacute

myeloidleukaemiaaftertransplantationintoSCIDmice1997BonnetDandcolleaguesidentifythefirstcancerstemcells,

theleukemiastemcell,insamplesfrompatientswithacute

myeloidleukemia2003

Al-HajjMandcolleaguesdescribetheidentificationofthe

breastcancerstemcells–thefirststemcelltobeidentified

fromasolidtumor2004

SinghSKandcolleaguesdescribetheidentificationofstemcellsfor

humanbraintumour2005

KimCFBandcolleaguesdescribetheidentificationofstemcellsfor

lungcancer2005

XinLandcolleaguesdescribetheidentificationofstemcellfor

prostatecancer2006

SzotekPPandcolleaguesdescribeovariancancersidepopulationdefinescellswithstemcells-likecharacteristics2006

SuetsuguAandcolleaguesdescribecharacterizationofCD133+

hepatocellularcarcinomacellsascancerstem/progenitorcells2007

Ricci-VitianiLandcolleaguesdescribetheidentificationofcolorectal

cancerStemcell2007

LiCandcolleaguesdescribetheidentificationofpancreaticcancer

Stemcells2007

PrinceMEandcolleaguesdescribeidentificationofasubpopulation

ofcellswithcancerstemcellspropertiesinheadandnecksquamous

cellcarcinoma2008

BussolatiBandcolleaguesdescribeidentificationofatumor-initiatingstem

cellpopulationinhumanrenalcarcinomas2009WangLandcolleaguesdescribecharacterizationofstemcellsattributesin

humanosteosarcomacelllinesMilestonesinaconceptofcancerasstemcellsdiseaseBiologicalcharactersofcancerstemcells

1.Theabilitiestoself-renewanddifferentiateintomultiplecelltypes2.Highoncogenicityandheterogeneitypromotingtumorigenesis.3.Multidrugandradiotherapyresistance4.Potentialofmicroinvasioninearlystageheterogeneityincancerisnotlimitedtodifferencesbetweendifferentpatients,butalsooccurswithinasinglepatientCancersCanBeHeterogeneousatManyDifferentLevel:Cancersfromthesameprimarysiteoforigincanhavedistinct,clinicallyrelevantbiologicdifferences(differenthistologicormolecularsubtypes)thatresultinveryheterogeneousbehaviorwithinonecancertype.Inaddition,withinonetumor,therecanbeintratumorheterogeneity,withdifferentclonesdominatingdifferentregionsofthesamecancer.Thisintratumorheterogeneitycanresultindivergentresponsestoselectivepressures,suchthatwithmetastaticprogression,differentclonescanpredominateindifferentmetastaticenvironments./oncology-journal/

heterogeneity-and-cancerSourcesofHeterogeneitywithinCancerThisheterogeneityamongcancercellsinthesamepatientcanariseinmultipleways:Themostwell-establishedmechanisminvolvesintrinsicdifferencesamongcancercellscausedbystochasticgenetic(Nowell,1976)orepigenetic(BaylinandJones,2011)changes(clonalevolution);Differencescanalsoariseamongcancercellsthroughextrinsicmechanismsinwhichdifferentmicroenvironmentswithinatumorconferphenotypicandfunctionaldifferencesuponcancercellsindifferentlocations(BissellandHines,2011);somecancersfollowastemcellmodelinwhichtumorigeniccancerstemcells‘‘differentiate’’intonontumorigeniccancercells,creatingahierarchicalorganization.Thedifferentiationofcancerstemcells

providesamechanismforgeneratingphenotypicandfunctionalheterogeneitybeyondtheheterogeneitythatcanbeattributedtoclonalevolutionorenvironmentaldifferences(Shackletonetal.,2009)SourcesofHeterogeneitywithinCancerMagee,etal.,2012HematopoieticStemCellNiches(AandB)Adulthematopoieticstemcells(HSCs)resideprimarilywithinbonemarrow.Bonemarrowisacomplexorgancontainingmanydifferenthematopoieticandnonhematopoieticcells.Shownin(B)isamagnifiedviewofthebonemarrowshowingsinusoids(red),bone(gray),andhematopoieticareas(lightred).Sinusoidsareoftenassociatedwithmegakaryocytes(purple),reticularcellsproducingthechemokineCXCL12(lightgreen),andmesenchymalprogenitors(white).Thebonesurfaceiscoveredbybone-resorbingosteoclasts(darkgreen)aswellasbone-liningcellsthatcandifferentiateintobone-formingosteoblasts.HSCs(blue)arefoundadjacenttosinusoidalbloodvessels(arrows)aswellasatorneartheendosteum(arrowhead).OsteoblastsandosteoclastselaboratefactorsthatregulateHSCmaintenanceandlocalization(Adamsetal.,2006).PerivascularreticularcellsandmesenchymalprogenitorshavealsobeenproposedtoelaboratefactorsthatregulateHSCmaintenance(Sacchettietal.,2007).(CandD)Low(C)andhigh(D)magnificationviewsofasectionthroughmousebonemarrowshowingaCD150+CD48CD41LineageHSC(whitearrowpointingtoredcell)adjacenttoasinusoid.ThisHSCisclosetotheendosteum(dottedline),butnotdetectablyincontactwithcellsliningbone.(B,bone;V,bloodvessel;orangecell,megakaryocyte;greencells,moredifferentiatedhematopoieticcells).CSCandnicheThecancerstemcellsnichemicroenvironmentscontainmultiplesourcesofcytokineproduction.Theyplayacrucialroleinthemaintenanceofcancerstemcellscharacteristicssuchaspluripotencyandself-renewal.Antonio.TargetingNotchtoTargetCancerStemCells.ClincancerRes.2010.Wnt/β-cateninpathwayHedgehogsignalpathwayNotchpathway上皮间质转化（epithelialmesenchymaltransition,EMT）

EMT指上皮细胞在特定的生理和病理情况下向间充质细胞转分化的现象，细胞极性消失，细胞骨架重排，迁移运动能力增强。EMT的生理或病理生理学意义EMT的信号调节通路四、以信号转导为靶的肿瘤治疗所谓靶向治疗(targetedtherapy)就是将靶向药物与对促进肿瘤形成和发展有重要作用的蛋白相结合，抑制这些蛋白的功能，从而减缓肿瘤细胞的增殖和侵袭转移等特性，达到治疗肿瘤的目的。肿瘤信号转移通路的研究

——使肿瘤靶向治疗成为可能抗信号转导治疗针对信号转导系统各组成部分受体激酶连接蛋白转录因子组合靶标/信号通路MechanismofActionofImatinibMesylate(STI-571;Gleevec)HER2过表达细胞Trastuzumab与HER2受体结合生长信号

被阻断抑制肿瘤

细胞生长Trastuzumab作用：

靶标抗癌药物就是针对分子靶点的抗癌药物，这类药物的针对性强，效果显著。改变了传统化疗药物对所有快速分裂的细胞全面打击的方式。这类药物的出现，标志着癌症治疗新时代的开始。研究热点集中在针对细胞表面靶标的单克隆抗体和信号转导抑制剂。五、常用的信号转导研究方法Proteinexpression Westernblot √ Proteinphosphorylation

Kinaseassay √DNA-proteininteraction

Gelshiftassay(Mobilityshiftassay) Reportergeneassay Chromatinimmunoprecipitation √Protein-proteininteraction Immunoprecipitation Two-hybridsystem √Blockingsignalingpathway Dominant-negativemutant Anti-senseoligonucleotide RNAinterference √ Ribozyme √ SmallmoleculeinhibitorsWesternBlotAnalysisKinaseassayChromatinImmunoprecipitation-ChIPYeasttwo-hybridsystem信号传导通路的阻断策略显性负性突变体

反义寡核苷酸技术

RNA干扰技术

核酶

脱氧核酶