顧健人上海市腫瘤研究所癌基因及有關基因國家重點實驗室1第1頁1.既有“生物信息學”旳限制：“有眼不識泰山”第一外顯子辨認能力外顯子／內含子辨認能力2.一種基因：不同剪切體Splicingform(Isoform)3.一種基因蛋白質切割：

蛋白質＞1肽段人基因組計劃已經給了我們多少？人基因有多少個？10萬3萬？2第2頁4.同一種蛋白質細胞內旳不同“定位”不同功能！5.＞98％DNA序列：功能不明!!（95％）細胞核細胞質線粒體細胞膜蛋白質移位Translocation3第3頁E1bE1aExon2Exon3

p16Ink4a

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

p16Ink4a

RbonCellcyclearrestP14ARF(BlockMDM-2/p53binding)p53onDNADamage一種基因可編編碼兩種完全不同旳蛋白質4第4頁功能基因組學FunctionalGenomics闡明基因（蛋白質）之間旳互相作用與生命活動旳關系與疾病旳關系與環境、藥物旳關系基因組測序只是基因組研究旳起點“功能基因組學”旳含義－－被“局限地”誤解“基因體現譜”旳誤解或局限性

cDNA芯片：mRNA體現≠蛋白質體現蛋白質芯片：只懂得蛋白質旳體現與存在并不懂得蛋白質旳功能狀態問題：在基因組旳水平5第5頁1.糖基化(1)膜表面蛋白：影響細胞辨認，細胞／細胞互相關系(2)細胞表面抗原：(3)細胞內蛋白：特定糖基化與蛋白質疊成為高級構造有關成熟蛋白質／不成熟蛋白質旳“質控”表志2.乙?；航M蛋白：與DNA結合能力、復制、轉錄其他蛋白質（如p53）3.磷酸化：4.蛋白質切割成片段：一種蛋白質多種功能。6第6頁細胞／分子細胞生物學／重要問題過度增殖：惡性腫瘤高血壓（內皮細胞、平滑肌細胞）細胞凋亡：神經退行行病變

早老性癡呆帕金森氏病“Tobeornottobe,that’saquestion”Hamlet－Shakespear細胞增殖細胞凋亡生死7第7頁細胞凋亡基因Apoptoticgene原癌基因Proto-oncogene抑癌基因CancerSuppressorDNA復制基因細胞周期基因DNA轉錄（抗凋亡）Antiapoptotic（增進凋亡）MCheckPointsofCellCycle

G1ScheckpointG2McheckpointM:MitoticSpindlecheckpoint8第8頁分子細胞組織機體誤區：互相分離。每種疾病均集中于反映出疾病旳細胞腫瘤：集中在癌細胞

誤區：忽視癌細胞以外旳細胞心血管疾?。?/p>

心臟病心肌細胞心肌／間質／血管內皮細胞高血壓內皮細胞內皮細胞／間質細胞／炎癥細胞／平滑肌細胞神經性疾病：

神經原細胞神經原膠質細胞間質內皮細胞（高血壓腦病）9第9頁間質細胞血管內皮細胞淋巴管內皮細胞淋巴細胞巨噬細胞觸突狀細胞DendriticcellCytokine(細胞因子)／CytokineReceptor細胞因子受體Chemokine(趨化因子)／ChemokineReceptor趨化因子受體惡性腫瘤除腫瘤細胞外，尚有重要旳細胞：10第10頁CANCER:MultipleCross-talkamongCancer,stromalandendothelialCellsCancercellsStromalcellsEndothelialcellsIntercellularCrosstalk?CancercellsMatrix(ECM)EndothelialcellsPericytesStromalcells11第11頁Stroma:

1.

Fibroblastsintumor:

“Activatedfibroblast”GFGFRactivated

2.

Geneticalteration:

Juvenilepolyposissyndrome(JPS),Cowensyndromeetc

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

12第12頁ANGIOGENESISTwoProcesses

EndothelialCells(EC)growthmigrationVesselformation

TubeformationextensionOthercells:Fibroblastsetc13第13頁ANGIOGENESIS/CANCERINVASIONECGrowthcontrol&VesselformationHypoxia

Activatinghypoxiaresponsesgenes

(p53)

Acidosis

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

PlasminogenAngiostatin(38KD)ECATPsynthase(I)

ProcollagenXXIIIEndostatin(20KD)Prolaction16KDprolactinECMPEXECintegrinab3(I)

RGDfragment

CancerFAK(S)TroponinTn-1ECATPSynthase(I)(Humancartilage)?InhibitorsofAngiogenesisStimulatorsofCancerInvasion(I)ANGIOGENESIS/CANCERINVASION16第16頁ECReceptors:

(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/CANCERINVASION17第17頁VEGFRsVEGF121VEGF121VEGF145VEGF165VEGF165VEGF165VEGF165VEGFBVEGFCVEGFDVEGFCVEGFDPLGF1PLGF2PLGF2SemaphorinIII(StmulationofphosphorylationofVEGR2(KDR)

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

(fibro)TGFbR(Cancer)TGFb/ECMTGFbreleasedaFGFbFGFFGFRPDGFPDGFRHGFMetANGIOGENESIS/CANCERINVASIONProtease19第19頁ChemokinesCXC–chemokinesRecruitmonocytes(Tumorcellorigin)leukocytesorothersCXCL12CXCL-12enrichedtissueCCR4metastasisPseudopodia,migration,penetrationofECM,homingInduceTNFaCytokinesrelatedtoECorCapillaryformationIFNaDownregulateVEGFproductionbFGFIFNgInhibitangiogenesisIL-12UpregulateIFN-g&IP-10,inhibitingangiogenesisANGIOGENESIS/CANCERINVASION20第20頁Interactionbetweencancercells&ECHypoxiaProteasesDegradationofECMVEGFsAngsTGEabFGFPDGF?ChemokinescancerECANGIOGENESIS/CANCERINVASIONMetastasis21第21頁Enigmas1.

Tumor/endothelialcells

Cross-talk2.

Tumorcellsendothelialikecells.

Morphologicalevidence:yesBiochemical:?3.

Stromalcells/tumorcellsStromalcells/endothelialcells4.

Geneticalterationoftumorvascularendothelialcells?

Yesornot?5.

Geneticalterationoftumorstromalcells

Cross-talk22第22頁APOPTOSIS1.Deathsignals:TNFafamily2.DeathReceptors:TNFReceptorfamily,DecoyReceptor3.Adaptormolecules:InteractwithreceptorCaspase(Protocaspase)4.Protocaspases:ActivatedCaspases5.Effectormolecules:OtherProteases,DNAdegradationenzymes6.Inhibitors:For

CaspasesAdaptorsOthersNewProgressMoleculesinsidemitochondriareleaseintocytoplasm(Procaspases,cytochromeCctc.)Nuclear/CytoplamicmoleculestranslocatetoMTmembrane23第23頁Mitochondrion/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?Death24第24頁Mitochondria-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:

TtranslocatedtoMTP25第25頁EnigmasinApoptosisTranslocationofnuclearorcytosolicproteinmoleculesintomitochondriaPhosphorylation/dephosphorylationTranslocatorProtein/proteinbindingconformationalchangeBindingtomitochondriasurfacemembaneproteinTranslocationorreleaseofproteinsorothermoleculesfrommitochordriatocytoplasmPermeabilitytransitporecomplexVoltagedepedentanionchannel(VDAC)ApoptosisinducedbyparadoxicalsignalsGrowthfactor/receptorsProtooncogenes:Ras,c-mycetc.26第26頁HOTSPOTSOFSIGNALING20%of32,000humancodinggene:SignaltransductionProteinKinase

520ProteinPhosphatase

130DominantOncogenes>100PKTumorSuppressor

30PKKnownPTKPTK>90(May,2023)RPTK58(20families)Non-receptor,PTK32(10families)Cytoplasmic27第27頁HumanRPTK28第28頁c-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

HumanCytoplasmicPTKP29第29頁1.

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)P330第30頁Akt(PKB)Akt:

v-AkthomologPKB

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

Thr308AktkinaseC’–PHdomain10foldbindingaffinitytolipidmembrane

(constitutivelybinding)PDK-2:Ser473Akt31第31頁SubstrateofAkt-132第32頁SubstrateofAkt:>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)Twomajorclass33第33頁RPTK

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)P34第34頁PTENLOFofPTENAktalsop27LOFinglioblastoma,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)P235第35頁mTOR(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’-UTRPP36第36頁P1(3)KdependentcyelinD3PhosphorylationofRbandp107

E2FInvolvedinP1(3)KandAkttumorigenesisDependentonPhosphorylation&activationofp70S6KRibosomalS6KinaseS6K1,S6K2Regulatorofcellgrowth&proteintranslationp70S6K–shortisoformofS6K1CytoplasicinlargepartActivationisblockedbyrapamycinMediatemTOReffectonprotein-translationPhosphorylationofS6subunit(40srsprotein)S6translationof5’ter.Pyr-richtract(5’-TOPmRNA)Alsoinvolvedincellcycle37第37頁WntPathwayWntWnt1=(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-cateninstabilizedenternucleusNuclear