突变体能够通过内源性的促进胰腺癌的转移演示文稿

突变体能够通过内源性的促进胰腺癌的转移演示文稿当前第1页\共有49页\编于星期四\6点（优选）突变体能够通过内源性的促进胰腺癌的转移当前第2页\共有49页\编于星期四\6点BackgroundpreventsenescencePromotemetastasismutantp53drugresistanceInduceapoptosisp53antineoplasticpreventangiogenesis当前第3页\共有49页\编于星期四\6点Background75%p53mutanthighlymetastaticpoorprognosisdrugresistance当前第4页\共有49页\编于星期四\6点BackgroundKPCcell:stablelyexpresssmallhaipinRNA,lostremaningp53wide-typealle KPflCcell:ap53nullcelllineKPCmice:develophighlymetastaticpancreaticcancerthatfaithfullymimicsthehumandisease当前第5页\共有49页\编于星期四\6点IdeasMutantp53maintainmetastaticphenotypetherelationshipbetweenMutantp53andPDGFRbonmetastasisPDGFRbmadiatesmetastasiswhenMutantp53wasdepletedimatinibcaninhibitmetastasisbytargetingPDGFRbPDGFRbcorrelateswithdisease-freesurvival

PDGFRbasaDownstreamMediatorofMutantp53当前第6页\共有49页\编于星期四\6点Methods1.WoundHealingandInvasionAssays2.RNASequencingandDataAnalysis3.ImmunostainingandMicroscopy4.qRT-PCR5.PDGFRbLuciferaseReporterAssay6.CoimmunoprecipitationandChromatinImmunoprecipitation7.ImmunohistochemistryandImmunofluorescence8.MouseStudies当前第7页\共有49页\编于星期四\6点Questionwhether

mutantp53isneededtosustainthemetastaticphenotypeand

howitisregulated？当前第8页\共有49页\编于星期四\6点RESULTS当前第9页\共有49页\编于星期四\6点KPC+sh.Ctrlcellexpressmutantp53划痕愈合率侵袭细胞数The

invasivenessdependon

mutantp53.Result1：SustainedExpressionofMutantp53IsRequiredforthe

InvasivePhenotypeofPancreaticCancerCells当前第10页\共有49页\编于星期四\6点转移瘤数量whethermutantp53expressionwasrequiredto

sustainthemetastaticpotentialofKPCcells?转移瘤荧光体视成像当前第11页\共有49页\编于星期四\6点Summary1theseresultsdemonstrate

that

mutantp53

cancontributeto

PDACinvasionandmetastasisandthatinhibitingitsactivitycanhaveanantimetastaticeffect当前第12页\共有49页\编于星期四\6点Questionhowmutantp53mediatestheinvasive

phenotypeofPDAC?当前第13页\共有49页\编于星期四\6点mutant

p53canaffecttheinvasivephenotypeofpancreaticcancer

cellsRNA测序，基因变化IPA分析Result2：TranscriptionalProfilingandFunctionalScreening

IdentifyPDGFRbasaDownstreamMediatorofMutant

p53inMurinePancreaticCancer当前第14页\共有49页\编于星期四\6点1：SLC40A12：SNED13：PDGFRbhypothesis：PDGFRbcouldaffectcellinvasionPDGFRb转录水平蛋白表达mutantp53敲除当前第15页\共有49页\编于星期四\6点细胞增殖不同亚型对侵袭的影响不同亚型蛋白表达情况当前第16页\共有49页\编于星期四\6点红色荧光蛋白绿色荧光蛋白两种细胞比值increasedPDGFRbdidnotconferaselectiveadvantagetotumorcellproliferation

当前第17页\共有49页\编于星期四\6点Summary2PDGFRbisnotrequired

fortheproliferationandtumorigenicpotentialofp53mutant

murinecancercellsbutspecificallyimpactstheirinvasive

potential.当前第18页\共有49页\编于星期四\6点QuestionIfthemutantp53-PDGFRbsignalingaxisacts

inhumancancercells？当前第19页\共有49页\编于星期四\6点PDGFRbmRNA水平四种人胰腺癌细胞（Mutp53敲除）不同种类肿瘤中Mutp53敲除Result3：PDGFRbMediates

Mutantp53ActioninHumanCancer

Cells当前第20页\共有49页\编于星期四\6点mutp53/PDGFRb分别敲除，A2.1细胞侵袭情况p53/PDGFRb敲除p53-/-细胞过表达PDGFRbinvasionp53-/-人胰腺癌细胞当前第21页\共有49页\编于星期四\6点Summary3upregulationofthePDGFRb

isimportantfortheactionofmutantp53inPDACandpossibly

othertumortypes.当前第22页\共有49页\编于星期四\6点p73can

repressthetranscriptionof

PDGFRbourKPCcells

expressedp73,butnotp63whetherthephysicalinteractionofmutantp53withp73mightimpairtheabilityofp73to

negativelyregulatetheexpressionofPDGFRb?mutantp53caninhibitp73andp63Question当前第23页\共有49页\编于星期四\6点KPflCPDGFRb荧光素酶报告基因免疫共沉淀Result4：

Mutantp53Disruptsthep73/NF-YComplextoMediate

PDGFRbExpressionandTumorCellInvasion当前第24页\共有49页\编于星期四\6点howp73repressesPDGFRb

transcription?Question当前第25页\共有49页\编于星期四\6点p73NF-YBPDGFRb序列×√NF-Y

bindingto

thePDGFRBpromoterp73/NF-Y

interactionhampersNF-YtobindandactivatethePDGFRB

promoterp73bindingwithNF-YBmutantp53canaffectp73/NF-YBPDGFRb结合定量×NF-YBNF-YANF-YC当前第26页\共有49页\编于星期四\6点whethertherepressiveactionofp73on

PDGFRbtranscriptionwasmediatedbyNF-Yandmodulated

bymutantp53?Question当前第27页\共有49页\编于星期四\6点KPflC细胞侵袭能力PDGFRb荧光素酶报告基因当前第28页\共有49页\编于星期四\6点KPflC细胞KPC侵袭能力修复当前第29页\共有49页\编于星期四\6点Summary4mutantp53promotesinvasion,inpart,dependsonitsabilityto

enhancePDGFRbexpressionthroughthedisruptionoftheinhibitoryp73/NF-Ycomplex当前第30页\共有49页\编于星期四\6点whetherPDGFRblevels

regulatemetastaticbehaviorofPDAC

cellsinmice?Question当前第31页\共有49页\编于星期四\6点肺转移肺组织病理切片Result5：

ModulationofPDGFRbExpressionLevelsMediatesthe

PhenotypicEffectsofMutantp53DepletionInVivo当前第32页\共有49页\编于星期四\6点转移灶大小免疫组化whetherpharmacologicinhibition

ofthePDGFRbpathwayrecapitulatestheeffectsofPDGFRbor

mutantp53depletion?当前第33页\共有49页\编于星期四\6点时效关系实验量效关系实验Crenolanib两种细胞IC50当前第34页\共有49页\编于星期四\6点whethercrenolanibcansuppressmetastasis?Crenolanib处理与DMSO处理荧光和免疫组化细胞凋亡当前第35页\共有49页\编于星期四\6点KPflC细胞pancreaticcancercellscanprovideasourceofPDGFligandthatcould

triggerautocrineactivationofPDGFRb条件培养当前第36页\共有49页\编于星期四\6点Summary5abrogationof

autocrineactivationsignalingofPDGFRb

leadstoasignificantreductionofinvasionandmetastasisdrivenbymutantp53.当前第37页\共有49页\编于星期四\6点whetherPDGFRbinhibitionpreventsthedevelopmentofmetastasisinKPCmice?

Question当前第38页\共有49页\编于星期四\6点量效关系抑制细胞增殖所需IC50体外KPC细胞侵袭实验Imatinib处理KPC的肺转移Result5：

ImatinibInhibitstheDevelopmentofMetastasesina

PDACMouseModelbyTargetingPDGFRb当前第39页\共有49页\编于星期四\6点thehighdiseaseburdeninthepancreaswastheprimarycauseofdeath肿瘤体积总体存活率当前第40页\共有49页\编于星期四\6点92%15%其他器官转移情况不同器官HE染色当前第41页\共有49页\编于星期四\6点DAPI,blue;CK8,red;pPDGFRb,green

imatinibwasabletoeffectively

inhibitPDGFRbactivityinprimarytumorsbasedonreduced

levelsofphospho-PDGFRb

inthetumorcellspPDGFRb强度imatinib对体内PDGFRb的影响当前第42页\共有49页\编于星期四\6点Summary6byinhibitingthekinase

activityofPDGFRb,imatinibsignificantlydiminishesthemetastaticpotentialofpancreaticcancercells.当前第43页\共有49页\编于星期四\6点whetherupregulationofPDGFRbiscorrelatedwithprognosisorwiththeclinicopathologicalcharacteristicsofPDACsinpatients.Question当前第44