RESEARCHOpenAccessAnoveltumorsuppressorproteinencodedbycircularAKT3RNAinhibitsglioblastomatumorigenicitybycompetingwithactivephosphoinositide-dependentKinase-1XinXia1,2†,XixiLi1,2†,FanyingLi1,2†,XujiaWu1,2,MaoleiZhang1,2,HuangkaiZhou1,2,NunuHuang1,2,XuesongYang1,2,FeizheXiao3,DaweiLiu4,LixuanYang1,2andNuZhang1,2*AbstractBackground:TheRTK/PI3K/AKTpathwayplayskeyrolesinthedevelopmentandprogressionofmanycancers,includingGBM.Asaregulatorymoleculeandapotentialdrugtarget,theoncogenicroleofAKThasbeensubstantiallystudied.ThreeisoformsofAKThavebeenidentified,includingAKT1,AKT2andAKT3,buttheirindividualfunctionsinGBMremaincontroversial.Moreover,itisnotknowniftherearemoreAKTalternativesplicingvariants.Methods:High-throughputRNAsequencingandquantitativereversetranscription-PCRwereusedtoidentifythedifferentiallyexpressedcircRNAsinGBMsamplesandinpairednormaltissues.HighthroughputRNAsequencingwasusedtoidentifycirc-AKT3regulatedsignalingpathways.Massspectrometry,westernblottingandimmunofluorescencestaininganalyseswereusedtovalidateAKT3-174aaexpression.ThetumorsuppressiveroleofAKT3-174aawasvalidatedinvitroandinvivo.ThecompetinginteractionbetweenAKT3-174aaandp-PDK1wasinvestigatedbymassspectrometryandimmunoprecipitationanalyses.Results:Circ-AKT3isapreviouslyuncharacterizedAKTtranscriptvariant.Circ-AKT3isexpressedatlowlevelsinGBMtissuescomparedwiththeexpressioninpairedadjacentnormalbraintissues.Circ-AKT3encodesa174aminoacid(aa)novelprotein,whichwenamedAKT3-174aa,byutilizingoverlappingstart-stopcodons.AKT3-174aaoverexpressiondecreasedthecellproliferation,radiationresistanceandinvivotumorigenicityofGBMcells,whiletheknockdownofcirc-AKT3enhancedthemalignantphenotypesofastrocytomacells.AKT3-174aacompetitivelyinteractswithphosphorylatedPDK1,reducesAKT-thr308phosphorylation,andplaysanegativeregulatoryroleinmodulatingthePI3K/AKTsignalintensity.Conclusions:OurdataindicatethattheimpairedcircRNAexpressionoftheAKT3genecontributestoGBMtumorigenesis,andourdatacorroboratethehypothesisthatrestor...
