肿瘤分子生物学的前沿问题

顾健人上海市肿瘤研究所癌基因及有关基因国家要点试验室11.既有“生物信息学”旳限制：“有眼不识泰山”第一外显子辨认能力外显子／内含子辨认能力2.一种基因：不同剪切体Splicingform(Isoform)3.一种基因蛋白质切割：

蛋白质＞1肽段人基因组计划已经给了我们多少？人基因有多少个？10万3万？24.同一种蛋白质细胞内旳不同“定位”不同功能！5.＞98％DNA序列：功能不明!!（95％）细胞核细胞质线粒体细胞膜蛋白质移位Translocation3E1bE1aExon2Exon3

p16Ink4a

P14ARF(human)P19ARF(mouse)CrossTalkbetwerenOncogenes&tumorsuppressorsOncogenicSignalsRas,c-myc,E2F

p16Ink4a

RbonCellcyclearrestP14ARF(BlockMDM-2/p53binding)p53onDNADamage一种基因可编编码两种完全不同旳蛋白质4功能基因组学FunctionalGenomics阐明基因（蛋白质）之间旳相互作用与生命活动旳关系与疾病旳关系与环境、药物旳关系基因组测序只是基因组研究旳起点“功能基因组学”旳含义－－被“局限地”误解“基因体现谱”旳误解或不足

cDNA芯片：mRNA体现≠蛋白质体现蛋白质芯片：只懂得蛋白质旳体现与存在并不懂得蛋白质旳功能状态问题：在基因组旳水平51.糖基化(1)膜表面蛋白：影响细胞辨认，细胞／细胞相互关系(2)细胞表面抗原：(3)细胞内蛋白：特定糖基化与蛋白质叠成为高级构造有关成熟蛋白质／不成熟蛋白质旳“质控”表志2.乙酰化：组蛋白：与DNA结合能力、复制、转录其他蛋白质（如p53）3.磷酸化：4.蛋白质切割成片段：一种蛋白质多种功能。6细胞／分子细胞生物学／主要问题过分增殖：恶性肿瘤高血压（内皮细胞、平滑肌细胞）细胞凋亡：神经退行行病变

早老性痴呆帕金森氏病“Tobeornottobe,that’saquestion”Hamlet－Shakespear细胞增殖细胞凋亡生死7细胞凋亡基因Apoptoticgene原癌基因Proto-oncogene抑癌基因CancerSuppressorDNA复制基因细胞周期基因DNA转录（抗凋亡）Antiapoptotic（增进凋亡）MCheckPointsofCellCycle

G1ScheckpointG2McheckpointM:MitoticSpindlecheckpoint8分子细胞组织机体误区：相互分离。每种疾病均集中于反应出疾病旳细胞肿瘤：集中在癌细胞

误区：忽视癌细胞以外旳细胞心血管疾病：

心脏病心肌细胞心肌／间质／血管内皮细胞高血压内皮细胞内皮细胞／间质细胞／炎症细胞／平滑肌细胞神经性疾病：

神经原细胞神经原胶质细胞间质内皮细胞（高血压脑病）9间质细胞血管内皮细胞淋巴管内皮细胞淋巴细胞巨噬细胞触突状细胞DendriticcellCytokine(细胞因子)／CytokineReceptor细胞因子受体Chemokine(趋化因子)／ChemokineReceptor趋化因子受体恶性肿瘤除肿瘤细胞外，还有主要旳细胞：10CANCER:MultipleCross-talkamongCancer,stromalandendothelialCellsCancercellsStromalcellsEndothelialcellsIntercellularCrosstalk?CancercellsMatrix(ECM)EndothelialcellsPericytesStromalcells11Stroma:

1.

Fibroblastsintumor:

“Activatedfibroblast”GFGFRactivated

2.

Geneticalteration:

Juvenilepolyposissyndrome(JPS),Cowensyndromeetc

DominanthereditaryStromalcellsabnormality,HamartomaHighriskforcoloncancerPTENandothers“Landscaper”“Stromaabnormalmicroenviroment”“Microenviromentoftumor-hostinterface”

12ANGIOGENESISTwoProcesses

EndothelialCells(EC)growthmigrationVesselformation

TubeformationextensionOthercells:Fibroblastsetc13ANGIOGENESIS/CANCERINVASIONECGrowthcontrol&VesselformationHypoxia

Activatinghypoxiaresponsesgenes

(p53)

Acidosis

VEGF-A(Cancer.EC)14Protease/matrix/Cancercellinvasion/AngiogenesisProteases:DegradationofECMandCellAssociatedProteinsMMPs(Marixmetalloproteinases),membrane-anchoredMMP2,MMP9,(CancerCell)SolubleMMP(fibro,EC)Adamalysin–relatedmembraneproteinasesBMP1metalloproteinasesTissueserineproteinasestPAUKThrombinPlasminuPA(EC,fibro.)uPAR(Cancer)InhibitorsPAITIMPs(TissueinhibitorofMMPs)PEX(DegradationproductofECMbyMMP2)15Proteasesfurthercreate

PlasminogenAngiostatin(38KD)ECATPsynthase(I)

ProcollagenXXIIIEndostatin(20KD)Prolaction16KDprolactinECMPEXECintegrinab3(I)

RGDfragment

CancerFAK(S)TroponinTn-1ECATPSynthase(I)(Humancartilage)?InhibitorsofAngiogenesisStimulatorsofCancerInvasion(I)ANGIOGENESIS/CANCERINVASION16ECReceptors:

(ECandCancerCell)

VEGFR1(Flt-1)VEGFR2(Flk-1,KDR)VEGFA-DPLGF

VEGFR3(Flt-4)

VEGFC,D

TIE1

?

TIE2

Ang1,23?4?

*Integrinab3*ECMPEX

ab5

(Laminin,tenasin)

CD36

Thrombospondin(ECMGlycoprotein)celladhesion,motility&growth

metallospondin

VascularTubuleformation*

EphB1

Recognizingsomeligandsorpartners

NHE3

?

ARNT

?

EPA-1

?Tenasoin:newECM.ANGIOGENESIS/CANCERINVASION17VEGFRsVEGF121VEGF121VEGF145VEGF165VEGF165VEGF165VEGF165VEGFBVEGFCVEGFDVEGFCVEGFDPLGF1PLGF2PLGF2SemaphorinIII(StmulationofphosphorylationofVEGR2(KDR)

VEGFR1VEGFR2VEGFR3Neurophilin1Neurophilin2(flt-1)(flk-1KDR)(flt4)Ligands:18ECGrowthFactors:SpecificGFs:VEGFA-DVEGFR2(KDR.Flk-1)PLGFVEGFR1(flt-1)Non-specificGFs(Local):Someofthem:autocrine(EC,CancerFibro,)TGFaEGFRTGFb

(fibro)TGFbR(Cancer)TGFb/ECMTGFbreleasedaFGFbFGFFGFRPDGFPDGFRHGFMetANGIOGENESIS/CANCERINVASIONProtease19ChemokinesCXC–chemokinesRecruitmonocytes(Tumorcellorigin)leukocytesorothersCXCL12CXCL-12enrichedtissueCCR4metastasisPseudopodia,migration,penetrationofECM,homingInduceTNFaCytokinesrelatedtoECorCapillaryformationIFNaDownregulateVEGFproductionbFGFIFNgInhibitangiogenesisIL-12UpregulateIFN-g&IP-10,inhibitingangiogenesisANGIOGENESIS/CANCERINVASION20Interactionbetweencancercells&ECHypoxiaProteasesDegradationofECMVEGFsAngsTGEabFGFPDGF?ChemokinescancerECANGIOGENESIS/CANCERINVASIONMetastasis21Enigmas1.

Tumor/endothelialcells

Cross-talk2.

Tumorcellsendothelialikecells.

Morphologicalevidence:yesBiochemical:?3.

Stromalcells/tumorcellsStromalcells/endothelialcells4.

Geneticalterationoftumorvascularendothelialcells?

Yesornot?5.

Geneticalterationoftumorstromalcells

Cross-talk22APOPTOSIS1.Deathsignals:TNFafamily2.DeathReceptors:TNFReceptorfamily,DecoyReceptor3.Adaptormolecules:InteractwithreceptorCaspase(Protocaspase)4.Protocaspases:ActivatedCaspases5.Effectormolecules:OtherProteases,DNAdegradationenzymes6.Inhibitors:For

CaspasesAdaptorsOthersNewProgressMoleculesinsidemitochondriareleaseintocytoplasm(Procaspases,cytochromeCctc.)Nuclear/CytoplamicmoleculestranslocatetoMTmembrane23Mitochondrion/CytoplasmCaspasesOtherSignalsInintermembranespaceProtocaspase293LatentAIFCytochrcmeCSmac/DIABLOLifeActivationofProtocaspase3,6,7ActivationofProtocaspase9OthertargestMitochondrionProtocaspase-2Protocaspase-3Protocaspase-9Apaf1CytochromecAIFOthers??(Smac/DIABLO)LifeorDeath?AlterationofMolecularCompartmentation(MitochondrionversusCytoplasm)OtherTargetsIntermembraneSpaceAIF

(Apoptosisinducingfactor)Science,397:387~91999Smac

(SecondMT-derivedactivatorofcaspase)Or

DIABLO

(DirectIAP-bindingproteinatLowpI)Bcl2guard?Death24Mitochondria-deathSignalIntegratorsC.Bremer&G.Koemer(Science.Aug.18.289:1150,2023)TranslocatetomitochondrinStimulation/inductionInhibition/blocktranslocation(Keepincytosol)PTPC:

PermeabilityTransitionPoreComplex(Baxetc.)Bcl2/Bax/BidpermeabilizetheouterMTmembraneuponinteractionwithPTPCBidmaybeindependentJNK:

(SAPK,stressactivatingproteinkinase)-inactivateBclXL

p53:

TranslocatedtoMT&interactedwithhsp70

PKC:

TtranslocatedtoMTP25EnigmasinApoptosisTranslocationofnuclearorcytosolicproteinmoleculesintomitochondriaPhosphorylation/dephosphorylationTranslocatorProtein/proteinbindingconformationalchangeBindingtomitochondriasurfacemembaneproteinTranslocationorreleaseofproteinsorothermoleculesfrommitochordriatocytoplasmPermeabilitytransitporecomplexVoltagedepedentanionchannel(VDAC)ApoptosisinducedbyparadoxicalsignalsGrowthfactor/receptorsProtooncogenes:Ras,c-mycetc.26HOTSPOTSOFSIGNALING20%of32,000humancodinggene:SignaltransductionProteinKinase

520ProteinPhosphatase

130DominantOncogenes>100PKTumorSuppressor

30PKKnownPTKPTK>90(May,2023)RPTK58(20families)Non-receptor,PTK32(10families)Cytoplasmic27HumanRPTK28c-src:

TruncatedmutationofC’toTyr530

autophosphorylation

relatedtoSTAT-3

incoloncancerc-abl:

CS9(9:22)abl/BCRinph1CSDNAdamageinducedapoptosis.ATMc-ablbindRbatG0/G1(releasedafterRBphosph.ofablpreventDNA-damageinducedablphosph.

PDGF

Inducecellmotility&adhesion(c-ablinvolvedin)

BCR/abl

Cyteplasm–Xnucleus

HumanCytoplasmicPTKP291.

ClassI

p110(catalytic)Adaptor/regulator

IA:RPTKactivatedIB:hetero-trimericGproteincoupledReceptoractivated.

ClassII

p1(3)k2.

Substrate:

ptdIns(4.5)P2ptdIns(3.4.5)p3SomeP1(3k)(I&III):+Ser/thrK

3.

Isoform

a,b,dofp110.Subunitp85a,p85b,p55gofadaptor4.

Bindingofproteintolipid

IAFVVEdomain

ptdIns(3)PPH(pleckstrinhomology)domain

PH(+)protein:3’-phospho-inositidedependentK(PDK1)(ser/thrPK)Akt(PKB)PI(3)K/Akt&mTOR/p7056k(rapamycintarget)PI(3k):heterodimerIAptdIns(3,4)P2ptdIns(3,4,5)P330Akt(PKB)Akt:

v-AkthomologPKB

a,b,gisoformsN’–PHdomainCentralkinasedomain–activationloop:Thr.308C’–Regulatorysite:Ser473PDK-1(3’-phospho-inositidedependentKinase1):

Thr308AktkinaseC’–PHdomain10foldbindingaffinitytolipidmembrane

(constitutivelybinding)PDK-2:Ser473Akt31SubstrateofAkt-132SubstrateofAkt:>13:RXRXXS/Tmotif

(thismotif:alsoforMAPKAPK-1&p7056K)Class1Class2Anti-apoptosisRegulatorofcellgrowthFKHR(ForkheadTA)

()eNOS

()Bad

()BRCA1

()CREB(cAMPResponseelement)

()GSK-3(GlycogensynthaseK)

()

(CyclinD)(b-catenin)PDE-3B(Phosphodiesterase–3B)

()p21WAF-1(

,viaFKHR)Twomajorclass33RPTK

activation

P1(3)k

PtdIns(3,4,5)P3PtdIns(3,4)P2InteractwithAKTAkt/PDK1Akttranslocatintomembrane

Thr308(loop)Ser473(Reg.Site)(PDK2?)PDK1/Aktactivation:AktPP

translocatetonucleiAKTPPPDK1

also－PKCisoforms,serumandglucoorticoidinducedK(GSK)PKCrelatedK(PRK)

p7056kp21activatedPK(PAK)P34PTENLOFofPTENAktalsop27LOFinglioblastoma,germcellcancer,breastcancerCancer&Pl(3)KIA/Akt

Aktexpression:pancreatic,ovariancancer

Akt//p65(mutantofp85aPl(3)k)

p110(Pl(3)K)amplification(ovariancancer)Pl(3)KIB(p110g)LOF:coloncancer(Bcl2,CDK.CyclinD)(3’-phosphoinositidephosphatase,TS)

ptdIns(3,4,5)P3ptd(3,4)P235mTOR(MammalianTargetofRapamycin)1.Conservedfamily:

Yeast:TOR1,TOR2,MEC1,TEL1,Rad3Drosophila:MEC41Mammal:mTOR,ATM,ATR(ATMrelated)TRAPP(transformation/transcriptionaldomain-associatedP)2.mTOR=FK506bindingP(FKBP)(human)

RapamycinassociatedP(FRAPhuman)RapamycinandFKBP12target-1(rat)3.mTOR

C’terminalKdomain(homologoustokinasedomainofPl(3)KandPl(4)Kser/theKactivityonly

mTORc-myc(inhibitedbymTORinhibitorrapamycin)

stat3(ser727)PKC;PKC4E-BPdissociation&releaseofelf-4Ecapdependentinitiationtranslationof5’-UTRPP36P1(3)KdependentcyelinD3PhosphorylationofRbandp107

E2FInvolvedinP1(3)KandAkttumorigenesisDependentonPhosphorylation&activationofp70S6KRibosomalS6KinaseS6K1,S6K2Regulatorofcellgrowth&proteintranslationp70S6K–shortisoformofS6K1CytoplasicinlargepartActivationisblockedbyrapamycinMediatemTOReffectonprotein-translationPhosphorylationofS6subunit(40srsprotein)S6translationof5’ter.Pyr-richtract(5’-TOPmRNA)Alsoinvolvedincellcycle37WntPathwayWntWnt1=(int-1,mouseMMTVintegrationactivatedgene)Porcupine:AgeneforwntsecretionPolytopicmembraneproteinRelatedtoacyltransferaseFrizzled(Fz)WntReceptorDally:Co-receptorofWntGlypican-typeheparansulfateproteoglycanGlycosyl-PtdInsmoietymembraneb-catenin:inabsenceofWnt

DestabilizedbyaproteincomplexDishevelled(Dsh)activatedbyWnt-Fzbinding

AxinAPCGSK-3b(Glycogensynthasekinase)

GSK-3b-b-catenin-b–catenindegradationWnt–Fzbinding

DshGSK-3b

b-cateninstabilizedenternucleu