Activation of PI3K/Akt pathway by CD133-p85 interaction promotes tumorigenic capacity of glioma stem cells

ActivationinteractionofofgliomastempromotesPI3K/Aktcells

tumorigenicpathwaybycapacityCD133-p85YuanyanWeia,1,YizhouJianga,1,FeiZoua,1,YingchaoLiub,1,ShanshanWanga,NuoXua,WenlongXuc,ChunhongCuia,YangXinga,YingLiua,BenjinCaoa,ChanjuanLiua,GuoqiangWua,HongAod,XiaobiaoZhangc,andJianhaiJianga,2

a

KeyLaboratoryofGlycoconjuatesResearch,MinistryofPublicHealth,DepartmentofBiochemistryandMolecularBiology,ShanghaiMedicalCollegeofFudanUniversity,Shanghai200032,People’sRepublicofChina;bDepartmentofNeurosurgery,ShandongProvincialHospital,ShandongUniversity,Jinan,Shandong250021,People’sRepublicofChina;cDivisionofNeurosurgery,ZhongshanHospital,FudanUniversity,Shanghai200032,People’sRepublicofChina;anddDepartmentofLaboratoryAnimalScience,FudanUniversity,Shanghai200032,People’sRepublicofChina

EditedbyGreggL.Semenza,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,MD,andapprovedMarch13,2013(receivedforreviewOctober2,2012)

Thebiologicalsigni canceofaknownnormalandcancerstemcellTodate,thewell-characterizedmechanismofAktactivationismarkerCD133remainselusive.Wenowdemonstratethattheactivatedbythephosphoinositide3-kinases(PI3Ks)(22,23).Thephosphorylationoftyrosine-828residueinCD133C-terminalcyto-PI3K/AktpathwaycanbeactivatedinawidespectrumofhumanplasmicdomainmediatesdirectinteractionbetweenCD133andcancersthroughtheinactivationofphosphataseandtensinho-phosphoinositide3-kinase(PI3K)85kDaregulatorysubunit(p85),mologtumorsuppressor,theactivationofreceptortyrosinekina-resultinginpreferentialactivationofPI3K/proteinkinaseB(Akt)ses,theampli cationofAktfamilymembers,orthemutationsofpathwayingliomastemcell(GSC)relativetomatchednonstemcell.thePI3Kcatalyticsubunit(24–26).Nonetheless,themechanismCD133knockdownpotentlyinhibitstheactivityofPI3K/Aktpath-regulatingthePI3K/Aktpathwaythatisspeci cincancerstemcellswaywithanaccompanyingreductionintheself-renewalandhasnotbeenadequatelyaddressed.Inthisstudy,weusedCD133+tumorigenicityofGSC.TheinhibitoryeffectsofCD133knockdowngliomastemcellmodeltoexplorethepossibilityofCD133asacouldbecompletelyrescuedbyexpressionofWTCD133,butnotitscomponentinregulatingthePI3K/Aktpathwayandtodeterminep85-bindingde cientY828Fmutant.AnalysisofgliomasamplesthebiologicalconsequenceofCD133-PI3Kinteraction.revealsthatCD133Y828phosphorylationleveliscorrelatedwithhistopathologicalgradeandoverlapswithAktactivation.OurResults

resultsidentifytheCD133/PI3K/Aktsignalingaxis,exploringtheCD133RegulatesAktSignaling.UsingtechniquesdescribedinthefundamentalroleofCD133ingliomastemcellbehavior.

Dirksgroup’soriginalreport rstvalidatingCD133asagliomaA

stemcell(GSC)cellsurfacemarker(6),weisolatedCD133+andccordingtothecancerstemcell(CSC)hypothesis,tumorsareCD133 cellsfromhumanglioblastomasamples(T21107,T21109,formedandmaintainedbyapopulationofundifferentiatedandT12179;cellsthatarecharacterizedbytheirabilityforself-renewalandto+pathologicaldataareshowninTableS1)(Fig.S1A).CD133tumorcellsshowedcharacteristicsconsistentwithcancerinducetumorigenesis(1,2).CriticaltoCSCresearchistheirstemcells:namely,neurosphereformation(Fig.S1B),expressionprospectiveidenti cationandisolationfromtumortissue.CD133ofstemcellmarkerNestin,andmultilineagedifferentiationwith(Prominin-1),a5-transmembranedomainglycoproteininitiallymarkersforastrocyte(GFAP),neuron(MAP2),oroligodendro-identi edinhumansasahematopoieticstemcellmarker(3,4),iscyte(O4)(Fig.S1+C).Inaninvivolimitingdilutiontumorforma-widelyusedasamarkerofcancerstemcellsinbraintumorsaswelltionassay,CD133tumorcellswerehighlytumorigenicinbrainsofasincoloncancer,hepatoma,andpancreaticcancer(5–9).How-immunocompromisedmicewithcharacteristicsofglioblastomainever,theutilityofCD133inde ningcancerstemcellshasbeenconcordancewithpreviousreport(6).CD133 cellsrarelyformedquestionedfollowingaseriesofarticles.Severalgroupshavedetectabletumorsevenwhenimplantedat1.0×105cellsperreportedthatCD133 glioblastoma(GBM)cellscanformtumorsmouse,exceptforoccasionaltumorsfromCD133 cellsderived(10–12).TheseeminglyelusiveroleofCD133inde ningcancerfromT12179sampleat1.0×105cells(Fig.S1D–F).Thus,ourdatastemcellsintheliteratureisanoutstandingdilemmaincancercon rmthatCD133+subpopulationsareenrichedforcharacter-researchtoday(13),raisingquestionsregardingthefunctionalisticsofgliomastemcells.

UsingthisCD133-basedselectionsystem,signi cancesandtheunderlyingpathwaysofCD133.

+wecomparedtheactivityofAktsignalingbetweenCD133gliomacellsandCD133–Increasing+evidencestronglysuggeststhefunctionalassociationgliomacells.AktachievesitsfullactivitythroughphosphorylationofCD133+cancerstemcellwithproteinkinaseB(Akt)signaling.atboththreonine308(T308)andserine473(S473)(27,28).Al-CD133tumorcellsderivedfromhepatoma,coloncancer,andthoughlevelsneuroblastomaconsistentlydisplayedincreasedphospho-Aktlevels oftotalAktproteinsweresimilarbetweenCD133+andCD133tumorcells,thephosphorylationsofAktonbothcomparedwithmatchedCD133 tumorcells(14–16).Indeed,theS473andT308weredramaticallyup-regulatedinCD133+

signi canceofactivatingAktsignalingincancerstemcellispro-vokedbytheknowninvolvementofAktsignalinginnormalstemcellbiologyandtumorigenesis(17–19)andbythedependenceofAuthorcontributions:Y.W.andJ.J.designedresearch;Y.W.,Y.J.,F.Z.,YingchaoLiu,S.W.,cancerstemcellonAktsignaling.ChemoresistanceinCD133+N.X.,W.X.,C.C.,Y.X.,YingLiu,B.C.,C.L.,G.W.,H.A.,X.Z.,andJ.J.performedresearch;J.J.hepatocarcinomastemcellsmaybeconferredbyactivationofAktcontributednewreagents/analytictools;Y.W.andJ.J.analyzeddata;andY.W.andJ.J.wrotethepaper.

(14).Inmousemedulloblastomamodels,AktregulatesthesurvivalTheauthorsdeclarenocon ictofinterest.

oftumorcellsintheperivascularnichebearingstemcellmarkersThisarticleisaPNASDirectSubmission.

(20).Furthermore,Aktinhibitioncouldproduceareductioninthe1Y.W.,Y.J.,F.Z.,andYingchaoLiucontributedequallytothiswork.

self-renewalandgrowthofCD133+cancerstemcellfromglioma2

andcoloncancer(16,21).However,themechanismsandsig-Towhomcorrespondenceshouldbeaddressed.E-mail:jianhaijiang@fudan.edu.cn.

ni cancesofAktactivationincancerstemcellremainunknown.

Thisarticlecontainssupportinginformationonlineatwww.1mpi.com.

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