Cancer Science. 2019;110:1573–1586.  | 1573wileyonlinelibrary.com/journal/cas

Received:27September2018  |  Revised:19February2019  |  Accepted:24February2019DOI:10.1111/cas.13984

O R I G I N A L A R T I C L E

Testis- specific protein, Y- linked 1 activates PI3K/AKT and RAS signaling pathways through suppressing IGFBP3 expression during tumor progression

Wenling Tu1 | Bo Yang2 | Xiangyou Leng1 | Xue Pei1 | Jinyan Xu1 | Mohan Liu1 | Qiang Dong2 | Dachang Tao1 | Yongjie Lu1 | Yunqiang Liu1  | Yuan Yang1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercialLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercialpurposes.©2019TheAuthors.Cancer SciencepublishedbyJohnWiley&SonsAustralia,LtdonbehalfofJapaneseCancerAssociation.

TuandYangcontributedequallytothiswork.

Abbreviations: APC, allophycocyanin; CT, cancer-testis; DEG, differentially expressed gene; GO, Gene Ontology; IGFBP3, insulin growth factor binding protein 3; KEGG, KyotoEncyclopediaofGenesandGenomes;LIHC,liverhepatocellularcarcinoma;LUAD,lungadenocarcinoma;MSY,male-specificregionoftheYchromosome;NAP,nucleosomeassemblingprotein;PI,propidiumiodide;RT-qPCR,real-timequantitativePCR;SET,suppressorofvariegation,enhancerofzeste,andtrithorax;TCGA,TheCancerGenomeAtlas;TSPY1,testis-spe-cificprotein,Y-linked1.

1DepartmentofMedicalGenetics,StateKeyLaboratoryofBiotherapy,WestChinaHospital,WestChinaMedicalSchool,SichuanUniversity,Chengdu,China2DepartmentofUrology,WestChinaHospital,SichuanUniversity,Chengdu,China

CorrespondenceYunqiangLiuandYuanYang,DepartmentofMedicalGenetics,WestChinaHospital,SichuanUniversity,Chengdu,China.Emails:yq_liu@scu.edu.cn(YL);yangyuan@scu.edu.cn(YY)

Funding informationTheNationalNaturalScienceFoundationofChina,Grant/AwardNumber:81370748,81773159 and 81871203; Sichuan Science andTechnologyProgram,Grant/AwardNumber:2018FZ0035

Thetestis-specificprotein,Y-linked1(TSPY1),anewlyrecognizedcancer/testisanti-gen,hasbeensuggestedtoacceleratetumorprogression.However,themechanismsunderlying TSPY1 cancer-related function remain limited. Bymining the RNA se-quencingdataof lungand livertumorsfromTheCancerGenomeAtlas,wefoundfrequentectopicexpressionofTSPY1inlungadenocarcinoma(LUAD)andliverhepa-tocellularcarcinoma(LIHC),andthemale-specificproteinwasassociatedwithhighermortality rate and worse overall survival in patients with LUAD and LIHC.OverexpressionofTSPY1promotescellproliferation,invasiveness,andcycletransi-tionandinhibitsapoptosis,whereasTSPY1knockdownhastheoppositeeffectsonthese cancer cell phenotypes. Transcriptomic analysis revealed the involvement of TSPY1inPI3K/AKTandRASsignalingpathwaysinbothLUADandLIHCcells,whichwas further confirmed by the increase in the levels of phosphorylated proteins in the PI3K-AKTandRASsignalingpathwaysinTSPY1-overexpressingcancercells,andbythesuppressionontheactivityof thesetwopathways inTSPY1-knockdowncells.Further investigation identified thatTSPY1coulddirectlybind to thepromoterofinsulingrowthfactorbindingprotein3(IGFBP3) to inhibit IGFBP3expressionandthatdownregulation of IGFBP3 increased the activity of PI3K/AKT/mTOR/BCL2 andRAS/RAF/MEK/ERK/JUNsignalinginLUADandLIHCcells.Takentogether,theob-servationsrevealanovelmechanismbywhichTSPY1couldcontributetothepro-gressionofLUADandLIHC.OurfindingisofimportanceforevaluatingthepotentialofTSPY1inimmunotherapyofmaletumorpatientswithTSPY1expression.

K E Y W O R D S

IGFBP3,PI3K/AKT,RAS,TSPY1,tumorprogression

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1  | INTRODUC TION

TSPY1 (testis-specific protein, Y-linked 1) is located in the male-specific region of the Y chromosome (MSY),1 representing thelargest andmosthomogenousprotein-coding tandemarray in thehuman genome.1,2 Previous studies have revealed that TSPY1, atestis-specificprotein, ispredominantlyexpressed inmature sper-matogonia and serves physiological functions in the proliferationand differentiation of spermatogonia during spermatogenesis.3,4 Remarkably,TSPY1isalsoinvolvedintheinitiationanddevelopmentof many tumors. TSPY1istheonlyMSYgenethatisdefinitelyrelatedtoaspecifictumor,gonadoblastoma.5-7Additionally,TSPY1isover-expressedinthemajorityofevaluatedtesticulargermcelltumors,8 andectopicallyactivated invarioussomaticcancers, includinghe-patocellular carcinoma, melanoma, and prostate cancer.9–11 All ofthisevidencesupportsanoncogenicroleofTSPY1ingermcellandsomatic tumors.

The predominant TSPY1 isoform is a 38-kDa phosphoproteinthat harbors a highly conserved SET/NAP domain.3 Its homologyto other proteins of the SET/NAP superfamily might suggest thefunctional diversity of TSPY1, including nucleosome assembly,transcription modulation, DNA replication, cell cycle control, andcell proliferation.12-14 To date, studies have evaluated severalmo-lecularmechanismsofthiscancer-testis(CT)proteinfunctions.Forexample,TSPY1potentiatescellproliferationandpromotesarapidtransitionfromG2toMphasethroughbindingtocyclinB1anden-hancingthekinaseactivityofcyclinB1/cyclin-dependentkinase1complex.15,16 It increases protein synthesis and gene transcriptionthroughinteractingwiththeeukaryotictranslationelongationfactor1A,17andexacerbatesthetransactivationofendogenousandrogenreceptorandactivatesanumberofgrowth-relatedandoncogeniccanonical pathways.18Recently,wereportedthatTSPY1promotescell proliferation through suppressing the ubiquitin-specific pepti-dase7-mediatedp53function.19Alltheseaccumulatingfindingsim-proveourknowledgeofthemechanismunderlyingTSPY1functionsunderphysiologicalorpathologicalconditions.However,ourunder-standingoftheclinicalsignificanceofTSPY1intheprogressionofthetumorsisstilllimited.Also,itisunknownwhethertherearesomecommonmechanismsunderlyingTSPY1oncogenicfunctionsindif-ferentkindsoftumor.ItisofimportancetoexploretheseissuesforevaluatingthepotentialoftheCTantigenasatumorimmunother-apytarget.

Lungandlivercancersarenowtheleadingcancerkillerworldwide.Inthepresentstudy,thecorrelationsbetweentheexpressionpatternofTSPY1andtheclinicalconsequencesinpatientswithlungorlivertumorswereexploredbyminingthedatasetsofTCGA.Transcriptomicanalysis was carried out to systematically investigate the TSPY1-influencedsignalingpathwaysandhubgenesinlungandlivertumorcells.Withthiswork,weobservedhighermortalityandworseoverallsurvivalinpatientswithTSPY1-expressedLUADandLIHCrelativetothosewithTSPY1-negativeLUADandLIHC.Importantly,werevealedthat TSPY1 could activatePI3K/AKT andRAS signaling through in-hibiting the transcription of IGFBP3. Our findings disclosed a novel

mechanismunderlyingthepromotionofcellproliferation,cellcycle-transition,invasiveness,andtheinhibitionofcellapoptosisbyTSPY1duringtumorprogression.

2  | MATERIAL S AND METHODS

2.1 | Bioinformatics analysis of TSPY1 expression profile and clinical features in male patients with lung and liver tumors

WeabstractedthemRNAexpressionprofilesofmalelungandlivertumorswith the clinical information from TCGA database (http://cancergenome.nih.gov, updated April 5, 2018). After normalizingmRNAexpressiondataobtainedfromRNAsequencing,thecancersthat showed recurrent ectopic expression of TSPY1 were deter-mined.Foreachofsuchcancers,theratioofTSPY1-positivecaseswas calculated and the difference in overall survival was assessed betweenpatientswithTSPY1-positiveand-negativecancerbythelog-ranktest.Thehazardratioswith95%confidenceintervalsandlog-rank P-values were calculated and displayed on the plot. ThemortalityratewascomparedbetweenTSPY1-positiveand-negativegroupsbytheχ2-testforeachtumortype.

2.2 | Transcriptomic analysis of TSPY1- overexpressing cells

Total RNA was isolated from TSPY1-overexpressing A549 andHepG2 cells usingTRIzol reagent (Invitrogen,Carlsbad,CA,USA).After determining the quality and concentration using an Agilent2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA),RNAs were sequenced in a HiSeq4000 (Illumina, San Diego, CA,USA)instrument.TriplicateRNAsamplesfromindependentgroupswereprepared for sequencing. Theprimarybioinformatic analysiswascarriedoutbyGenedenovoBiotechnology.(Guangzhou,China).Gene Ontology analysis was visualized using the Database forAnnotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/).GeneRatiowascalculatedbythepercentageofDEGsthatmatchaspecificGOterminthetotalDEGs.KyotoEncyclopediaof Genes and Genomes pathway enrichment analysis was under-taken using the KEGG Orthology-based Annotation System 2.0(KOBAS 2.0, http://kobas.cbi.pku.edu.cn). The protein-protein in-teractionnetworkwasanalyzedusingtheCytoscapesoftwarefromtheSTRINGdatabase (http://string-db.org/).Thehubgenes intheDEGswere identifiedaccording toa combinedconditions that in-cluded:(i)ahighconnectivitydegree(>3);(ii)involvementinoneoftheenrichedpathways; and (iii) regulationon importantbiologicalphenotypes.

2.3 | Reverse transcription and RT- qPCR

TotalRNAsfromcellswerereversetranscribedintocDNAsusinga RevertAid First-Strand cDNA Synthesis Kit (Thermo FisherScientific,Waltham,MA, USA). The RT-qPCRswere carried out

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usingSYBRPremixExTaqII(TaKaRaBio,Dalian,China)inaBio-Rad iCycler RT-qPCR Detection System (Bio-Rad, Berkeley, CA,USA).Eachassaywascarriedout in triplicate.GAPDHwasusedasan internalcontrol.TheRT-qPCRprimersfortargetgenesarelisted in Table S1.

2.4 | Western blot analysis

Cellswereharvestedinlysisbuffer(Bioteke,Beijing,China)witha protease inhibitor cocktail (Roche, Basel, Switzerland). Proteinconcentrations were determined using a BCA protein assay kit(Bioteke), and equal amounts of protein were loaded into SDS-PAGEforwesternblotting.Antibodiesusedinthisstudyarepre-sented in Table S2.

2.5 | Plasmid construction

The full-length cDNA encoding FLAG-tagged TSPY1 was synthe-sized and cloned into pcDNA3.1 (+) vector (Invitrogen) andpLVX-IRES-ZsGreen1 vector (Clontech, Mountain View, CA, USA). SixIGFBP3promoter fragmentswithdifferent lengths (TableS1)wereamplifiedandclonedintothepGL3-Basicluciferasereportervector(Promega,Madison,WI,USA).TSPY1-specificshRNA(shTSPY1)andIGFBP3-specificshRNA(shIGFBP3)weresynthesizedandinsertedintothepLKO.1vector(Addgene,Cambridge,MA,USA).TheTSPY1-specific siRNA (siTSPY1) and IGFBP3-specific siRNA (siIGFBP3)were synthesizedbyRiboBio (Guangzhou,China). Their target se-quencesarelistedinTableS1.

2.6 | Cell culture and lentivirus infection

Humancell lines293T (embryonickidney),A549 (lungadenocar-cinoma),HepG2(hepatocellularcarcinoma),LCLC-103H(largecelllung carcinoma), andMHCC97H (hepatocellular carcinoma)werecultured in DMEM and 1640 medium supplemented with 10%FBSand1%penicillin-streptomycininahumidifiedincubatorwith37°Cand5%CO2. The lentivirus system was composed of 3 vec-tors: pMD2G (VSV-G envelope) (Addgene), pSPAX2 (backbone)(Addgene), and pLVX-IRES-ZsGreen1 (stably expressed targetgene)orpLKO.1(shRNAtointerferethetargetgene).The3vectorsweretransfectedinto293TcellsusingajetPRIMEtransfectionkit(Polyplus,Illkirch,France).After48hours,viralsupernatantswerecollected,centrifuged,andfilteredthrough0.45-μmPVDFmem-branes.Thentheviralsupernatantswereusedtoinfectcells.After72hours,RT-qPCRsandwesternblotassayswereundertakentodetect the expression levels of TSPY1 and IGFBP3.Additionally,the lentivirus-infected cell clones of TSPY1-overexpressing or-knockdown and IGFBP3-knockdown cells were selected usingpuromycin. The siRNAs were transfected into the cells using ajetPRIME transfection kit (Polyplus). At 48hours after transfec-tion,totalproteinswereextractedfromthecellsforwesternblotassays.

2.7 | Dual- luciferase reporter assays

ADual-LuciferaseReporterAssaykit(Promega)wasusedfordetec-tionof luciferaseactivity. Inbrief,6plasmids,pGL3-P1,pGL3-P2,pGL3-P3, pGL3-P4, pGL3-P5, and pGL3-P6, were separately co-transfectedwith FLAG-TSPY1 or the negative control pcDNA3.1-FLAG plasmids into 293T cells. After 48hours, the luciferaseactivityofcelllysateswasdetectedaccordingtothemanufacturer'sprotocols.

2.8 | Chromatin immunoprecipitation assays

Chromatin immunoprecipitation assays were carried out using aMagna ChIP kit (Millipore, Temecula, CA, USA) according to themanufacturer'sprotocols.Briefly,cellswerefixedwith1%formal-dehyde for 10minutes, and cell lysates were sheared by sonica-tionin1%SDSlysisbuffertogeneratechromatinfragments.Then1%of theoptimally sheared chromatinwas retained as a positivecontrol “inputDNA” in thesubsequentPCRs.Theremainingchro-matin was immunoprecipitated with 2 μganti-FLAGAbsspecifictoFLAG-TSPY1.Antibody-protein-DNAcomplexeswereprecipitatedwith protein A/G magnetic beads. The DNA eluted and purifiedwassubjectedtoPCRusingprimersspecificforthetargetregionsof the IGFBP3promoter.TheprimersforPCRarelistedinTableS1.ImmunoglobulinGwasusedasanegativecontrol.

2.9 | Cell proliferation and colony formation assays

ThecellproliferationratewasmeasuredwiththeCCK-8(Beyotime,Shanghai, China) according to the manufacturer's instructions.Briefly,cellswereseeded into96-wellplatesatadensityof3000cellsperwell.At indicatedtimepoints,CCK-8solutionwasaddedto each well. After 2hours of incubation at 37°C, 5% CO2, theabsorbanceofeachwellwasmeasuredat450nmusingamicroplatereader.Eachgroupwasmeasuredintriplicate.Therelativeprolifera-tionwasdeterminedasafoldchangethatwascalculatedusingtheabsorbanceofeachwell.Theresultswerenormalizedbythevalueof a control.

Forthecolonyformationassay,300cellswereplatedinto6-wellplates and incubated for 2weeks. Colonies were then fixed withmethanol and stained with crystal violet solution when the colonies weresufficiently largeforvisualization.Thestainedcolonieswerecountedandphotographed.Eachgroupwas tested independentlyin triplicate.

2.10 | Cell cycle and apoptosis assays

Forthecellcycleassay,cellswereresuspendedandfixedwith70%prechilled ethanol overnight at 4°C. The fixed cells were washedwith coldPBSand treatedwithRNaseA solution (250μg/mL) for30minutes. Finally, the cells were stainedwith PI (Sigma-Aldrich,St. Louis,MO,USA) andassessedona flowcytometer (Beckman,

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Krefeld, Germany). The measured results were analyzed usingModFitsoftware(VeritySoftwareHouse,Topsham,ME,USA).

CellapoptosiswasdetectedusinganannexinV-APC/PIapoptosisdetection kit (BDPharmacy, Franklin Lakes,NJ,USA) according tothemanufacturer'sprotocol.Briefly,theindicatedcellswerewashedwithcoldPBSandresuspendedwithstainingbuffer.Avolumeof5μLannexinV-APCand1μLPIwereaddedtoatotalof100μLcellsus-pension.Themixwasincubatedatroomtemperaturefor15minutesandthensubjectedtoflowcytometryanalysis.ApoptoticcellswereidentifiedasbothannexinV-APC+/PI−andannexinV-APC+/PI+.

2.11 | Cell invasion assay

Cellswereculturedinserum-freemediumfor24hoursandplatedintoupperTranswellfilterchamberscoatedwithMatrigel(Corning,Corning,NY,USA).Mediumwith10%FBSwasaddedtothelowerchamberasa chemoattractant to drive cell movement and incubated for 12 hours. Invaded cells on the undersides of the membrane were fixed withmethanolandstainedwithcrystalviolet.Cellswerephotographedandcountedunderamicroscope.Eachassaywascarriedoutintriplicate.

2.12 | Statistical analysis

Student'sttestwasusedtocomparetheexperimentalgroupswithSPSSsoftware(version17.0;IBM,Chicago,IL,USA).Numericaldatawere reported as the mean ± SD. P < .05 was considered statistically significant.

3  | RESULTS

3.1 | Ectopic expression of TSPY1 significantly affects the mortality rate and overall survival of patients with LUAD and LIHC

AfterextractingtheRNAsequencingdatafromTCGA,weanalyzedthe TSPY1 expression profile in different kinds of lung and liver

tumors,eachofwhichcontainedmorethan50sampleswithrelevantclinical information. Our results showed frequent ectopic expres-sion of TSPY1mRNA in lungsquamouscell carcinoma,LUAD,andLIHC(FigureS1A).ForexploringtheclinicalsignificanceofTSPY1intumorprogression,wefurtherinvestigatedtheeffectofTSPY1ex-pressiononmortalityrateandoverallsurvival.Significantly,resultsshowedahighermortalityrateinthepatientswithTSPY1-positiveLIHC relative to those with TSPY1-negative LIHC (FigureS1B).Moreover, we observed that patients with TSPY1-positive LUADorLIHChadworseoverallsurvivalthanthosewithTSPY1-negativetumor (Figure1A,B). However, the influence of TSPY1 on overallsurvivalwasnotfoundinlungsquamouscellcarcinoma(Figure1C),which might have resulted from the limited number of samples.Collectively, these findings provided evidence to suggest that theectopicexpressionofTSPY1promotedtumorprogression.

3.2 | Overexpression of TSPY1 promotes proliferation, invasiveness, and cycle transition and inhibits apoptosis of cancer cells

To investigate the impact of TSPY1during tumorprogression,weconstructed2celllines,A549andHepG2,inwhichTSPY1wasover-expressedexogenously(Figure2A).Thenweexaminedthecellchar-actersofproliferation,invasiveness,cycletransition,andapoptosisinthe2TSPY1-overexpressingcell lines.WeobservedthatTSPY1overexpression resulted in enhanced cell proliferation (Figure2B),colonyformation(Figure2C),andinvasiveability(Figure2D)intheA549andHepG2cells.WealsofoundthatTSPY1overexpressionincreasedthecellratiooftheG1/G0 phase and decreased that of the G2/Mphase(Figure2E),indicatingthatTSPY1promotedthephasetransitionofG2toMintheA549andHepG2cells.Additionally,weobserved that TSPY1 overexpression reduced the percentage ofapoptoticcells intheA549andHepG2cells (Figure2F).Takento-gether,thesefindingssuggestedthatTSPY1promotedtheprolifera-tion, invasiveness,andcycletransitionand inhibitedtheapoptosisofcancercells,andthatthe2TSPY1-overexpressingcell linescan

F IGURE  1 Poorprognosisassociatedwiththeectopicexpressionoftestis-specificprotein,Y-linked1(TSPY1)inpatientswithlungadenocarcinomaandliverhepatocellularcarcinoma.Foreachtumor,theprognosiswascomparedbetweenpatientswithandwithouttheectopicexpressionofTSPY1incancertissue,intheformofasurvivalcurve.DeteriorationofoverallsurvivalwasobservedinpatientswithTSPY1-positivelungadenocarcinoma(A)andliverhepatocellularcarcinoma(B)relativetoTSPY1-negative(log-ranktest,α=0.05:lungadenocarcinoma,P = .0203;liverhepatocellularcarcinoma,P = .0050).Suchaninfluencewasnotidentifiedinlungsquamouscellcarcinoma(C).HR,hazardratio

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beusedtoexplorethepotentialmechanismthatenablesTSPY1topromotecancerprogression.

3.3 | Transcriptomic analysis reveals a potential influence of TSPY1 on the function of both PI3K- AKT and RAS signaling pathways

To investigate the common mechanism by which TSPY1 contrib-utestotheprogressionandcellphenotypesofLUADandLIHC,weexplored thechangesof thegeneexpressionprofiles in theA549and HepG2 cells when TSPY1 was overexpressed through RNA

sequencing. The sequencing data from this study have been sub-mittedtotheNCBIshortreadarchiveportalunderaccessionnum-ber SRP152744; raw sequence data were submitted to the NCBIBio-project PRJNA479692. We observed that 501 genes wereupregulated and 208 genes were downregulated in the TSPY1-overexpressing A549 cells, and that 117 genes were upregulatedand 222 geneswere downregulated in the TSPY1-overexpressingHepG2cells(FigureS2).Sixteenupregulatedgenesandninedown-regulated genes were found in both TSPY1-overexpressing celllines(FigureS2).Then,weundertookGOclassificationtofunction-ally annotate theDEGs.We obtained a total of 294 and 289GO

F IGURE  2 Promotionofcellproliferation,cellcycletransition,cellinvasiveness,andinhibitionofcellapoptosisbytestis-specificprotein,Y-linked1(TSPY1)inA549lungadenocarcinomacellsandHepG2liverhepatocellularcarcinomacells.A,WesternblotanalysisshowedthatTSPY1wasexogenouslyoverexpressedinA549andHepG2cells.B,CCK-8assaysshowedthatTSPY1overexpressionenhancedtheproliferationofA549andHepG2cells.Therelativeproliferationispresentedasthefoldchange,calculatedbasedonabsorbanceandnormalizedtoacontrolvalue.C,ColonyformationassaysshowedthatTSPY1overexpressionincreasedthecolonynumbersinA549andHepG2cells.Originalmagnification,×1.D,TranswellassaysshowedthatTSPY1overexpressionacceleratedinvasionofA549andHepG2cells.Originalmagnification,×400.E,CellcycleassaysshowedthatTSPY1overexpressionpromotesG2/Mphasetransitioninthecellcycle,andashorterphasetransitionwasfoundinA549andHepG2cells.F,CellapoptosisassaysshowedthatTSPY1overexpressioninhibitsapoptosisofA549andHepG2cells.Dataarepresentedasthemean±SD(n=3,*P < .05)

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assignmentsintheTSPY1-overexpressingA549andHepG2cells,re-spectively;approximately60%comprisedbiologicalprocesses,19%comprisedmolecularfunction,and21%comprisedcellularcompo-nentsinbothcells(Figure3A).Inthebiologicalprocesscategory,theDEGswere involved in“responsetostimulus” (A549,DEGpropor-tion42.74%;HepG2,47.55%),“localization”(A549,36.54%;HepG2,38.11%), “cellular developmental process” (A549, 31.48%;HepG2,38.11%), “celldifferentiation” (A549,30.67%;HepG2,36.71%)and“single-organism developmental process” (A549, 33.44%; HepG2,40.56%).Inthecategoryofmolecularfunction,mostDEGsplayrolesin“binding”(A549,58.75%;HepG2,50.35%)and“catalyticactivity”(A549, 25.25%; HepG2, 29.43%). As for the cellular component,a large percentage of DEGs were associated with the categories

“cytoplasm” (A549, 60.73%; HepG2, 63.34%), “membrane” (A549,33.64%; HepG2, 28.94%), and “nucleus” (A549, 30.9%; HepG2,33.12%).Furthermore,weusedtheKEGGpathwayenrichmentanal-ysistoidentifypotentialbiologicalpathwaysinwhichTSPY1mightbeinvolved.TheKEGGanalysisshowedthatTSPY1mightaffectthefunctionofseveralsignalingpathways;onlythePI3K-AKTandRASsignalingpathwayswerefoundtohaveanassociationwithTSPY1inbothA549andHepG2cells(Figure3B).Therefore,wefurtherinves-tigatedtheinfluenceofTSPY1onthefunctionofthesetwosignalingpathwaysintheA549andHepG2cells.

In addition, we undertook a protein-protein interaction net-work analysis to identify the most significant DEG “hub genes”whoseexpression is regulatedbyTSPY1during tumorprogression

F IGURE  3 RNAsequencing-basedtranscriptomicevidencefortheinvolvementoftestis-specificprotein,Y-linked1(TSPY1)inimportantbiologicalfunctionsandsignalingpathwaysinA549andHepG2cells.A,Differentiallyexpressedgenes(DEGs)wereobtainedbyRNAsequencingofA549andHepG2cellswithandwithoutTSPY1overexpression.GeneOntology(GO)analysisshowedthatthefunctionsoftheDEGscouldbeassociatedwith3mainGOcategoriesincludebiologicalprocess,cellularcomponent,andmolecularfunction.EachbarrepresentstherelativeabundanceofDEGsclassifiedundereachcategory.B,KyotoEncyclopediaofGenesandGenomespathwayenrichmentanalysisshowedthatTSPY1couldbeassociatedwiththefunctionofPI3K/AKTandRASsignalingpathwaysinTSPY1-overexpressingA549andHepG2cells

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accordingtotheSTRINGdatabase(FiguresS3andS4).Weobtained11hubgenesintheA549cellsand6hubgenesintheHepG2cells(Figure4A).WethenverifiedthemRNAlevelalterationofthesehubgenesusingRT-qPCRanalysiswhichshowedahighconsistencywiththeRNAsequencingresults(Figure4B-D).Interestingly,the4genesJUN,BCL2,RUNX2,andHCFC1wereobservedinbothcells.Ofthem,JUN and BCL2weredetectedtohavethehighestdegreeofconnec-tivityinthenetworkthatweconstructedusingtheselected13hubgenes (Figure4E).Given thatBCL2 and JUNare important down-streammoleculesofPI3K/AKTandRASsignalingpathways,respec-tively,20,21 we postulated that TSPY1 increased the expression ofBCL2 and JUNthroughupregulatingtheactivitiesofthesepathways.

3.4 | Overexpression of TSPY1 promotes activation of PI3K/AKT/mTOR/BCL2 and RAS/RAF/MEK/ERK/JUN signaling pathways

To validate the influence of TSPY1 on PI3K/AKT andRAS signal-ingpathways,weanalyzedtherelevantproteinlevelchangesintheTSPY1-overexpressingA549andHepG2cells(Figures5andS5).AsshowninFigure5A,weobservedthatthephosphorylatedproteins

p-AKTandp-mTORinthePI3K-AKTsignalingpathwayweresignifi-cantly increased in both TSPY1-overexpressing A549 and HepG2cells and that the upregulation ofBCL2proteinwas also verified.In addition,we found thatTSPY1contributed to theupregulationof p-RAF1, p-MEK1/2, and p-ERK1/2 in the RAS signaling path-wayinbothA549andHepG2cellsandthattheJUNprotein levelwas consequently increased (Figures5B and S5). Considering thesignificance of PI3K/AKT/mTOR/BCL2 and RAS/RAF/MEK/ERK/JUNsignalingpathwaysincellproliferation,cycle,invasiveness,andapoptosis,22-26wepropose that TSPY1 affects cell biological phe-notypes probably through a cross-talk network that involves themolecules with functional regulation of both PI3K/AKT and RASsignalingpathways.

3.5 | Inhibition of IGFBP3 expression might be a potential way of TSPY1 promoting the activation of PI3K/AKT and RAS signaling pathways

Among the network containing 13 hub genes, we found thatIGFBP3 and RUNX2 closely connected with BCL2 and JUN (Figure4E). Considering that the downregulation of IGFBP3 and

F IGURE  4  IdentificationofthehubgenesJUN and BCL2involvedinthePI3K/AKTandRASsignalingpathways,respectively,intestis-specificprotein,Y-linked1(TSPY1)-overexpressingA549andHepG2cells.A,Atotalof13differentiallyexpressedgenes(11inA549and6inHepG2cells)wereclassifiedashubgenes,ofwhich4(JUN,BCL2,RUNX2,andHCFC1)werefoundinbothcells.B,RNAsequencingheatmapshowingdifferentexpressionofthe13hubgenesinTSPY1-overexpressingA549andHepG2cells,relativetocontrols.C,D,Expressionlevelchangesofthe13hubgeneswerevalidatedbyreal-timequantitativePCRanalysisinTSPY1-overexpressingA549andHepG2cellsrelativetocontrols.Dataarepresentedasthemean±SD(n=3,*P < .05).E,Protein-proteininteractionnetworkanalysisofthehubgenesshowed that JUN and BCL2werelocatedinthecenterofthenetworkwiththehighestdegreeofconnectivityandthattherewere6hubgenes(SERPINE1,RUNX2,BCL2L11,KIT,CXCR4,andIGFBP3)withpotentialconnectionwithbothBCL2 and JUN

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the upregulation of RUNX2 expression were confirmed in bothTSPY1-overexpressingcells,wesuspectedthatIGFBP3 and RUNX2 mightalsobe involvedintheTSPY-regulatedPI3K/AKTandRASsignalingpathways.Asatranscriptionfactorthatplaysanimpor-tantrole intheregulationofcellproliferation,cellcycle,celldif-ferentiation,andapoptosis,RUNX2hasbeen reported tobe thedownstreammolecule of the PI3K/AKT andRAS signaling path-ways as its transcription-regulation activity is dependent on thephosphorylation obtained from p-AKT and p-ERK.27 Contrarily,IGFBP3 has been suggested to function as an important up-stream regulator of both PI3K/AKT andRAS signaling pathwaysas IGFBP3,actingasan insulin-likegrowth factor1bindingpro-tein,caninhibitthebioactivityofinsulin-likegrowthfactor1thatactivatesPI3K/AKTandMAPKsignaling.28Inaddition,apreviousreporthasshownthatectopicTSPY1activationand lowIGFBP3expressionwerepresentedsimultaneouslyinmanyhepatocellularcarcinoma cases.9Thus,wehypothesizedthatTSPY1suppressedIGFBP3expression,whichresulted inpromotedactivityofPI3K/AKTandRASsignalingpathways.

Toverify thishypothesis,we first confirmed the suppressionof TSPY1on the IGFBP3protein level inA549 andHepG2 cells(Figure6A). Thenweknockeddown IGFBP3expression inA549and HepG2 cells, using both shRNA and siRNA targeted to theIGFBP3gene(Figures6BandS6),andobservedthatthereductionof IGFBP3was associatedwith the increase of p-AKT, p-mTOR,BCL2,p-RAF,p-MEK,p-ERK,andJUN(Figures6B,CandS6).TheseresultssupportedthatthefunctionofTSPY1inhibitingIGFBP3ex-pressioncanactivatethepathwaysofPI3K/AKTandRAS.

3.6 | Transcriptional activity of IGFBP3 suppressed by TSPY1 directly binding to the promoter of IGFBP3

To investigate whether TSPY1 directly binds to the promoter ofIGFBP3toregulateitstranscription,weconstructed6luciferasere-portervectorscontainingthepromoterfragmentsdifferent insizes

(Figure7A). After co-transfecting the constructs with a TSPY1-expressedvectorindividually,wefoundthat,comparedtoacontrolvector, the relative luciferaseactivitiesof4 fragmentsweresignifi-cantlydecreased(Figure7B).Correspondingly,thepotentialbindingregionofTSPY1intheIGFBP3promotermightbelimitedfrom−272to−172bp.TofurtherverifythedirectbindingofTSPY1totheIGFBP3 promoter,weundertookChIPassaysusingananti-FLAGAbtoprecip-itateFLAG-TSPY1proteininthechromatinderivedfromtheTSPY1-overexpressingA549andHepG2cells.Thenvarious regionsof theIGFBP3promoterwereamplifiedwiththeimmunoprecipitatedDNA(Figure7C,D),usingseveralpairsofspecificprimers(Figure7A),andthePCRproductswereverifiedbyDNAsequencing.Theresultssug-gestedthatTSPY1occupiedonthefragmentsfrom−388to−205bpandfrom−225to−78bpintheIGFBP3promoter(Figure7C,D).Takentogether,thesefindingssuggestedthatTSPY1mightbindtothere-gionfrom−225to−205bpoftheIGFBP3 promoter to suppress the transcriptional activity of IGFBP3,decreasingIGFBP3expression.

3.7 | Knockdown of TSPY1 presents an opposite effect on the biological phenotypes and the activity of PI3K/AKT and RAS signaling pathways in both LCLC- 103H and MHCC97H cells

ConsideringthatTSPY1islessexpressedinbothA549andHepG2cells, we checked the effect on the cell phenotypes when theTSPY1levelwasdownregulatedinlungcarcinomacell lineLCLC-103H and liver hepatocellular carcinoma cell line MHCC97H inwhich cells TSPY1 is constitutively expressed (Figure8A). Asexpected, the cell proliferationand invasivenesswere clearly in-hibited(Figure8B-D),thetransitionfromG2 toMphasewasob-structed(Figure8E),andapoptosiswasaccelerated(Figure8F)intheLCLC-103HandMHCC97Hcells inwhich theTSPY1expres-sionwasinhibitedbyspecialshRNAandsiRNA.Theseresultsindi-catedthatthetargetedblockofTSPY1expressionwouldimpedetumorprogression.

F IGURE  5 PromotionoftheactivationofPI3K/AKTandRASsignalingpathwaysbytestis-specificprotein,Y-linked1(TSPY1)inA549andHepG2cells.A,Westernblotanalysisshowshigherlevelsof3keyproteins(p-AKT,p-mTOR,andBCL2)ofthePI3K/AKTsignalingpathwayinTSPY1-overexpressingA549andHepG2cellsrelativetocontrols.B,Westernblotanalysisshowshigherlevelsof4keyproteins(p-RAF-1,p-MEK1/2,p-ERK1/2andJUN)oftheRASsignalingpathwayinTSPY1-overexpressingA549andHepG2cellsrelativetocontrols

     |  1581TU eT al.

We then investigated the levels of proteins involved in the sig-nalingpathwaysofPI3K/AKTandRASintheTSPY1-downregulatedLCLC-103HandMHCC97Hcells,andobservedthatthedecreaseofTSPY1 expression upregulated the IGFBP3 expression (Figures9AandS7),andcontrarilyreducedtheproteinlevelsofp-AKT,p-mTOR,BCL2,p-RAF,p-MEK,p-ERK,andJUN(Figures9andS7).Thesere-sultsfurtherconfirmedthepositiveinfluenceofTSPY1ontheacti-vationofPI3K/AKTandRASsignalingpathways through inhibitingIGFBP3expression.

4  | DISCUSSION

The ectopic activation of TSPY1 is frequentlyobserved invarioussomatic cancers, and somepotentialmechanismsassociatedwiththe involvement of TSPY1 in tumor progression have been re-ported.15-19Inthepresentstudy,wepresentedanovelmechanismunderlyingthepromotionofTSPY1onIGFBP3-mediatedtumorpro-gression(Figure10).WeidentifiedthatTSPY1promotedtheactiva-tionofbothPI3K/AKT/mTOR/BCL2andRAS/RAF/MEK/ERK/JUN

F IGURE  6 Knockdownofinsulingrowthfactorbindingprotein3(IGFBP3)promotestheactivationofPI3K/AKTandRASpathwaysinA549andHepG2cells.A,Westernblotanalysisconfirmedthattestis-specificprotein,Y-linked1(TSPY1)reducedtheendogenousIGFBP3expressioninA549andHepG2cells.B,Western blot analysis shows decreased IGFBP3expressionwascorrelatedwiththeincreasedlevelof3keyproteins(p-AKT,p-mTOR,andBCL2)ofthePI3K/AKTsignalingpathwayinA549andHepG2cells.C,Westernblotanalysisshowedhigherlevelsof4keyproteins(p-RAF-1,p-MEK1/2,p-ERK1/2,andJUN)oftheRASsignalingpathwayinA549andHepG2cellswithIGFBP3knockdown,relativetocontrols(shNCandsiNC)

1582  |     TU eT al.

signalingpathways,andthispromotiondependedontheinhibitionof TSPY1 on IGFBP3 expression. The PI3K/AKT and RAS signal-ingpathwaysarekeysignaltransductioncomponentsintheregu-lationofcellgrowth,celldifferentiation,cellcycle,apoptosis,andmetastasis,22-26 and recent reports show thatderegulatedactiva-tionofthe2signalingsisfrequentlyobservedinmanycancers.29-31 Therefore,ourfindingsprovidedapotentialcommonmechanismofTSPY1'sfunctionasanoncogeneinthetumorprocess.

PreviousstudieshaveshownthatIGFBP3,atumorsuppressor,inhibitscellproliferation,promotesapoptosis,andreducesgrowthinvariouscancers,includingprostatecancer,32colorectalcancer,33 breastcancer,34 and ovarian endometrioid carcinoma.35 Recent re-ports also showed that the low levels of IGFBP3were correlatedwithpoorprognosisforpatientswithhepatocellularcarcinoma36 and that Igfbp3-nullmice experienced increased lung tumorburden.37 Theseobservationsstronglysuggestthesignificanceof IGFBP3intumorigenesis.Inthepresentstudy,wefoundtheinhibitoryeffectof TSPY1on IGFBP3 transcriptional activity by binding to IGFBP3 promoter and the following activation of PI3K/AKT and RAS sig-nalingpathways.ThesefindingsprovideevidencethatTSPY1isanupstreammodulatorofIGFBP3andtheectopicactivationofTSPY1disruptsthesuppressingabilityofIGFBP3ontumorprogression.

Consideredatranscriptionregulator,TSPY1wasfoundtoupreg-ulateitstranscriptionalactivitybybindingtotheexon-1ofitsowngene.38 It alsopromotes the transcriptionof itshomologousgeneTSPYL5 by binding to the TSPYL5 promoter.19 Surprisingly, in thisstudy,wefoundthatTSPY1hadanegativeeffectonthetranscrip-tion of IGFBP3, indicatingthatthetransactivatingabilityofTSPY1dependsontheavailabilityofotherspecificcofactors.Similarly,B-myb,anothertranscriptionalfactor,alsopossessesdualcharactersin the transcription regulation activities of repression and activa-tion.39,40ArecentreportalsoshowedthatB-myb,likeTSPY1,sup-pressed the IGFBP3 expression and upregulated the proliferationandmigrationofnon-small-cell lungcancercells.41GiventhatSETandNAP1familyproteinsplaymultiple functions, suchashistonechaperones,42 the transcriptional regulation ability of enhancer orsuppressormightdependonadditionalregulatoryfactorsthatcangenerate a transcriptional complex with TSPY1. However, thesetranscriptionalcomplexescontainingTSPY1need tobeverified infutureinvestigations.

Sex disparities in incidence and progression are frequentlyidentified in various human diseases.43 The risk of greater inci-dence, higher mortality rate, and lower survival are observed inmale patients relative to female patients for 32 of 36 cancer types

F IGURE  7 MechanismofactionunderlyingthesuppressionofthetranscriptionalactivityofIGFBP3bytestis-specificprotein,Y-linked1(TSPY1).A,SchematicoftheIGFBP3promoterdepictingthelocationofIGFBP3promoterconstructs(P1-P6)andChIP-PCRprimers(PCR1-PCR4).B,LuciferasereporterassaysshowedthatTSPY1coulddecreasetheluciferaseactivityof4IGFBP3promoterconstructs(P1-P4)in293Tcells.C,D,ChIP-PCRassaysrevealedTSPY1directlyoccupiedthebindingsitefrom−388to−205bpandfrom−225to−78bpoftheIGFBP3promoterinA549(C)andHepG2(D)cells.Moreover,ChIP-qPCRanalysisshowedhigheraTSPY1levelintheIGFBP3 promoter when comparedtoIgG.Dataarepresentedasthemean±SD(n=3,*P < .05)

     |  1583TU eT al.

studied.44,45Themechanismsresponsibleforthegenderdifferencesincancerdevelopmentandprognosisremainlargelyunknown.Themost significantgenetic traitofmen is geneson theirY chromo-some,which could provide a reason for suchmale preference incancer.ArecentstudyhasrevealedthatthelossoftheYchromo-some in the peripheral blood is closely associated with shorter sur-vivalandhigherincidenceriskofcancerinmales.46Additionally,theectopic expression of MSY-linked genes, including TSPY1, RBMY,and VCY, was observed in various male-biased somatic cancers,particularlyinliverandlungcancer.9,47-49Theseobservationssug-gestthattheYchromosomemightbeinvolvedinthedevelopment,

progression, and outcomes of cancers in amale-specificmanner.Inthecurrentstudy,weconfirmedthattheectopicexpressionofTSPY1wasassociatedwithhighermortalityandworseoverallsur-vivalprobabilityinmalepatientswithLUADorLIHC,whichfurtherprovidedevidencetosuggest thatTSPY1mightcontributetothesexdisparitiesinsomecancers,particularlyLUADandLIHC.

Cancer/testisantigensare important forcancerdiagnosisandimmunotherapy due to their tumor-restricted expression patternandhigh immunogenicity. Several clinical trials assessedCTanti-gens, suchasMAGE-A3andNYESO-1, asvaccine therapy inpa-tientswithlung,prostate,andovariancancersandmelanoma.50-53

F IGURE  8 Knockdownoftestis-specificprotein,Y-linked1(TSPY1)inhibitscellproliferation,cellcycletransition,andcellinvasivenessandpromotescellapoptosisinLCLC-103HlungcarcinomaandMHCC97Hlivercarcinomacells.A,WesternblotanalysisshowsdecreasedendogenousTSPY1inLCLC-103HandMHCC97HcellsaftertransfectionwiththelentiviralTSPY1shRNAvector.B,CCK-8assaysshowthattheknockdownofTSPY1inhibitedtheproliferationofLCLC-103HandMHCC97Hcells.Relativeproliferationispresentedasthefoldchange,calculatedbasedonabsorbanceandnormalizedtoacontrolvalue.C,ColonyformationassaysshowthattheknockdownofTSPY1decreasedthecolonynumbersofLCLC-103HandMHCC97Hcells.Originalmagnification,×1.D,TranswellassaysshowthattheknockdownofTSPY1couldimpairinvasionofLCLC-103HandMHCC97Hcells.Originalmagnification,×400.E,CellcycleassaysshowthattheknockdownofTSPY1couldextendG2/Mphase.F,CellapoptosisassaysshowthattheknockdownofTSPY1couldpromoteapoptosisofLCLC-103HandMHCC97Hcells.Dataarepresentedasthemean±SD(n=3,*P < .05).shNC,ScrambledshRNAvectoractingasanegativecontrol

1584  |     TU eT al.

Cancer-testisgenesaregenerallylocatedontheXchromosome,54 rarelyontheYchromosome.However, it is importantto identifyYchromosome-linkedCTgenes,particularlyforthediagnosisandimmunotherapyofmalepatientswithcancers.Inthepresentstudy,we observed that TSPY1was frequently ectopically activated indifferentmalesomatictumorspecimensandidentifiedasignificantcorrelationbetweenTSPY1activation,highermortality,andworseoverall survival probability inmale patientswith LUAD or LIHC.Theclinical findings, togetherwith thepotentialofTSPY1 incellproliferation,cellinvasion,cellcycletransition,andcellapoptosis,suggest that TSPY1 acting as aCT antigen could be a target fordiagnosisandimmunotherapyinmalepatientswithsomecancers.

Inconclusion,wepresentedacomprehensivesetofclinicalandexperimentalevidenceestablishingTSPY1asanoncogenicfactorthat

facilitatestumorprogressionandcausespoorprognosisinmalepa-tientswithLUADorLIHC.OurfindingsrevealedthatTSPY1stronglypromotestheactivationofthePI3K/AKTandRASsignalingpathwaysthroughsuppressingIGFBP3genetranscription,providinganovelex-planationforthecontributionofTSPY1totumorprogression.

ACKNOWLEDG MENTS

This work was supported by Grants from the National NaturalScience Foundation of China (Nos. 81370748, 81773159 and81871203) and Sichuan Science and Technology Program (No.2018FZ0035). We are grateful to Professor Yun-Fai Chris Lau(UniversityofCalifornia)forprovidingthemousemonoclonalAbagainstTSPY1.

F IGURE  9 Knockdownoftestis-specificprotein,Y-linked1(TSPY1)suppressestheactivationofPI3K/AKTandRASpathwaysinLCLC-103HandMHCC97Hcells.A,Westernblotanalysisshowsincreasedinsulingrowthfactorbindingprotein3(IGFBP3)expressionandlowerlevelsof3keyproteins(p-AKT,p-mTOR,andBCL2)ofthePI3K/AKTsignalingpathwayinLCLC-103HandMHCC97HcellswithTSPY1knockdown,relativetocontrols(shNCandsiNC).B,Western blot analysis shows lower levels of4keyproteins(p-RAF-1,p-MEK1/2,p-ERK1/2,andJUN)oftheRASsignalingpathwayinLCLC-103HandMHCC97HcellswithTSPY1knockdown,relativetocontrols(shNCandsiNC)

     |  1585TU eT al.

CONFLIC T OF INTERE S T

The authors declare that they have no conflicts of interest.

ORCID

Yunqiang Liu https://orcid.org/0000-0001-7691-7630

YuanYang https://orcid.org/0000-0002-9206-0312

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:TuW,YangB,LengX,etal.Testis-specificprotein,Y-linked1activatesPI3K/AKTandRASsignalingpathwaysthroughsuppressingIGFBP3expressionduringtumorprogression.Cancer Sci. 2019;110:1573–1586. https://doi.org/10.1111/cas.13984