RESEARCHOpenAccessTheTGFβ-miR-499a-SHKBP1pathwayinducesresistancetoEGFRinhibitorsinosteosarcomacancerstemcell-likecellsTianWang†,DexingWang†,LianZhang,PingYang,JingWang,QiLiu,FeiYanandFengLin*AbstractBackground/aims:Anovelparadigmintumorbiologysuggeststhatosteosarcoma(OS)chemo-resistanceisdrivenbyosteosarcomastemcell-likecells(OSCs).Asthesensitivityofonlyafewtumorstoepidermalgrowthfactorreceptor(EGFR)tyrosinekinaseinhibitors(TKIs)canbeexplainedbythepresenceofEGFRtyrosinekinase(TK)domainmutations,thereisaneedtoelucidatemechanismsofresistancetoEGFR-targetedtherapiesinOSthatdonotharborTKsensitizingmutationstodevelopnewstrategiestocircumventresistancetoEGFRinhibitors.Methods:AsameasureofthecharactersofOSCs,serum-freecultivation,cellviabilitytestwitherlotinib,andserialtransplantationinvivowasused.Westernblotassayswereusedtodetecttheassociationbetweenerlotinibresistanceandtransforminggrowthfactorbeta(TGFβ)-inducedepithelial-to-mesenchymaltransition(EMT)progression.ByusingTaqManqPCRmiRNAarray,onlinepredictionsoftware,luciferasereporterassaysandwesternblotanalysis,wefurtherelucidatedthemechanisms.Results:Here,CD166+cellsarefoundin10outof10tumorsamples.WecharacterizethatCD166+cellsfromprimaryOStissuesbearhallmarksofOSCsanderlotinib-resistance.TGFβ-inducedEMT-associatedkinaseswitchisdemonstratedtopromoteerlotinib-resistanceofCD166+OSCs.FurthermechanismsstudyshowthatTGFβ-inducedEMTdecreasesmiR-499aexpressionthroughthedirectbindingofSnail1/Zeb1tomiR-499apromoter.OverexpressionofmiR-499ainCD166+OSCsinhibitsTGFβ-inducederlotinib-resistanceinvitroandinvivo.SHKBP1,thedirecttargetofmiR-499a,regulatesEGFRactivityreductionoccurringconcomitantlywithaTGFβ-inducedEMT-associatedkinaseswitchtoanAKT-activatedEGFR-independentstate.TGFβ-inducedactivationofAKTco-optsanincreasedSHKBP1expression,whichfurtherregulatesEGFRactivity.Inclinic,theratiooftheexpressionlevelsofSHKBP1andmiR-499aishighlycorrelatedwithEMTandresistancetoerlotinib.Conclusion:TGFβ–miR-499a–SHKBP1networkorchestra...
