Millipore6070s

JournalofCancer2020,Vol.
11http://www.
jcancer.
org6070JournalofCancer2020;11(20):6070-6080.
doi:10.
7150/jca.
44367ResearchPaperZIC2promotescancerstemcelltraitsviaup-regulatingOCT4expressioninlungadenocarcinomacellsWangWei-Hua1*,ZhouNing1*,ChenQian2andJiangDao-Wen11.
Departmentofthoracicsurgery,MinhangHospital,FudanUniversity,Shanghai201100,China.
2.
Departmentofgeneralsurgery,MinhangHospital,FudanUniversity,Shanghai201100,China.
*Theseauthorscontributedequallytothismanuscript.
Correspondingauthors:JiangDao-Wen,Departmentofthoracicsurgery,MinhangHospital,FudanUniversity,Shanghai201100,China.
Tel.
:8621-64175590,Fax:8621-64175590;E-mail:jiang_daowen1@163.
com;ortoChenQian,Departmentofthoracicsurgery,MinhangHospital,FudanUniversity,Shanghai201100,China,Tel:8621-64175590,Fax:8621-64175590;E-mail:chenqian2010@sohu.
com.
Theauthor(s).
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(https://creativecommons.
org/licenses/by/4.
0/).
Seehttp://ivyspring.
com/termsforfulltermsandconditions.
Received:2020.
01.
29;Accepted:2020.
06.
29;Published:2020.
08.
19AbstractBackground:Accumulatingevidencehasrevealedtheimportanceofcancerstemcells(CSCs)inself-renewalandchemoresistance.
PreviousstudiesreportedhighexpressionofZIC2wascloselyassociatedwithtumorigenesisandCSCtraits.
However,theroleofZIC2asacrucialfactorforregulatingCSCpropertiesinlungadenocarcinoma(LAC)remainselusive.
Methods:RT-PCRandWBassaywereemployedtoassessZIC2expressionin20LACtumortissuesandthematchednon-canceroustissues.
TheroleofZIC2inLACCSCwereanalyzedbyevaluationofCSC-relatedmarkersexpressionandspheroidformationinvitro.
CisplatinandpaclitaxelresistancecapacitieswereevaluatedbyCCK8assay,colonyformationassay,andflowcytometryanalysis.
SubcutaneousNOD/SCIDmicemodelsweregeneratedtoassessinvivoCSCfeatures.
Results:HighexpressionofZIC2wasfoundinLACtumortissuesandindicatedapooroverallsurvivalinLACpatients.
ZIC2upregulatedanarrayofCSCs-relatedgenes,includingEpCAM,OCT4,SOX2,NANOG,C-MycandBmi-1.
KnockdownofZIC2inhibitedsphere-formingcapacityanddecreasedcisplatinandpaclitaxelresistance.
However,overexpressionofZIC2achievedoppositeeffects.
Mechanically,ZIC2actsupstreamofOCT4topromoteitsexpression,resultinginenhancementofCSCtraitsinLAC.
Conclusion:OurresultsdemonstratedthatZIC2wascrucialforpromotingCSCtraitsinLACcells,andservedasapotentialbiomarkerforpredictingprognosis.
TheZIC2-OCT4networkwillfacilitatetheevaluationofthepotentialtherapeuticefficacyofchemotherapyandpredictpatientsensitivitytotreatment.
Keywords:lungadenocarcinoma;cancerstemcells;ZIC2;OCT4activationIntroductionLungcanceristhemajorcauseofcancer-relateddeathsworldwide[1-3].
Lungadenocarcinoma(LAC)islistedasthemostcommonsubtypesoflungcancer,accountingforalmost40%[4-6].
DistantmetastasisandchemoresistanceareprimarilyattributedtoshortsurvivalforLACpatients[7].
Thecancerstemcells(CSCs)aredefinedasararesubsetofcancercellsandpossessesabilitytodifferentiateandself-renew[8].
Theyarealsoresponsiblefortumorgrowth,cancerousrecurrence,metastasisandchemoresistance[9].
Therefore,understandingoftherolesandmechanismscriticaltolungCSCgenerationandexpansionwouldimprovetheoverallsurvivalratesinLACpatients.
Transcriptionfactor-mediatedexpressionregulationisoneofthecriticalregulatorymechanismscontributingtoCSCstraits[10].
OCT4,NANOGandSOX2havebeenidentifiedasthecorepluripotentTFsinembryonicstemcells,whichplayimportantrolesinestablishingandmaintainingthepluripotencyofstemIvyspringInternationalPublisherJournalofCancer2020,Vol.
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OCT4,amemberofthePOUfamily,exertsafundamentalroleincancerstemcellself-renewalandmultipledifferentiationpotential[14].
PreviousstudiesrevealedthatOCT4waspreferentiallyexpressedinLACstemcellsandshowedcloselycorrelationwiththegenerationandexpansionofLACCSCs[15-17].
Additionally,OCT4iscriticaltoinducedrugresistanceinLACpatients[17].
However,whyOCT4exhibitedhighexpressioninLACstemcellsremainedunclear.
Thus,itisessentialtoexploretheregulatorymechanismsofOCT4expression,whichwouldcontributetounderstandthetumorgenesisanddrugresistanceinLACandobtainmorepotentialtherapeuticbenefitsinclinic.
Thezincfingerfamilymember2(ZIC2)functionsasatranscriptionalregulatorandhasbeenreportedtobehighlyexpressedinsolidtumors[18].
Importantly,highexpressionofZIC2iscloselyassociatedwithtumorigenesisandself-renewalofcancercells[19].
AccumulatingevidenceindicatedZIC2maybeinvolvedinlungcancerprogression[20,21].
Interestingly,ZIC2-dependentOCT4expressionwasinvolvedinregulatingCSCtraitsinlivercancer[19].
However,theroleofZIC2inLACwasstillunknown.
Here,wefoundthatZIC2wasupregulatedinLACtissuesandindicatessignificantlypoorerprognosis.
Additionally,ZIC2greatlyenhancedsphere-forming,proliferationandchemoresistancecapacitiesofLACcells.
Importantly,werevealedthatZIC2wasakeyupstreamregulatorygeneofOCT4,whichsustainsthestemnessofLACCSCs.
MaterialsandMethodsHumantissuesamplesPairedperi-tumorandtumortissuespecimenswereobtainedfrom20LACpatientsintheDepartmentofthoracicsurgery,MinhangHospital,FudanUniversity,forsubsequentexperiments.
Apathologicaldiagnosiswasmadeaccordingtothehistologyoftumorspecimensorbiopsyandwasexaminedbyexperiencedpathologists.
Allparticipantsinvolvedinthisstudyprovidedinformedconsentbeforethecommencementofthestudy.
CelllinesNormallungcellsHBECandhumanLACcelllinesA549,NCI-H1975,PC9,NCI-H1650,NCI-H23,NCI-H1299werepurchasedfromCellBankofTypeCultureCollectionofChineseAcademyofSciences(Shanghai,China).
CellswereculturedinDulbecco'sModifiedEagleMedium(DMEM)(Gibco,USA)containing10%fetalbovineserum(FBS)(Gibco,USA)and100units/mlofpenicillinand100g/mlofstreptomycin(Gibco,USA)inahumidified37°Cincubatorwith5%CO2.
AccordingtotheexpressionofZIC2inlivercelllines,wechosetheZIC2-highexpressingLACcelllineNCI-H1299toperformtheloss-of-functionexperiments.
TheZIC2low-expressingLACcelllineNCI-H1975wasusedtoperformthegain-of-functionexperiments.
EstablishmentofstablecelllinesStablecelllineswereestablishedaspreviouslydescribed[18].
TwoshRNAsequencesspecificallyagainstZIC2(shZIC2#1,shZIC2#2)andcontrol-shRNAwereused.
TwodistinctshRNAHuSH29-mershRNAconstructsagainstZIC2inpGFP-V-RSvec-torwerepurchasedfromMerdobioTechnologies(Shanghai,China).
TheproductionoflentiviralparticlesandsubsequentinfectionofNCI-H1299andNCI-H1975cellswereperformed.
Positivecellswereselectedwithpuromycin(Clontech,MountainView,CA,USA)for10days.
RT-PCRmRNAwasextractedaccordingtostandardprotocolsprovidedbyTRIzol(Invitrogen,USA).
ThetotalmRNAwasreversedtocDNAfollowedbythePrimerScriptTMRTreagentKitwithgDNAEraser(PerfectRealTime)(TaKaRa,China).
Quantitativereal-timePCRwasperformedwithEvaGreen2*qPCRMasterMix(Invitrogen,USA)inaCFX96TMReal-TimePCRSystem(BioRad,USA).
RT-qPCRwasperformedintriplicate.
TherelativelevelsofmRNAwerenormalizedtothoseofGAPDH.
TheprimersequencesusedarelistedinSupplementalTable1.
WesternblotWesternblotwasperformedaspreviouslydescribed.
Indetails,culturedcellswerelysedinRIPAlysisbuffer(Beyotime,China)supplementedwithcocktailproteaseinhibitor(Roche,Switzerland).
ProteinswereseparatedbySDS-PAGEandweretransferredontopolyvinylidenedifluoridemembranes(Millipore,USA).
Themembraneswereblockedin5%nonfatmilksolutionfor1hat37°Candwereincubatedwithprimaryantibodiesat4°Covernight,followedbyincubationwithHRP-conjugatedsecondaryantibodies(Beyotime,China)for1h.
ThesignalswerevisualizedusingtheBeyoECLPluskit(Beyotime,China).
TheantibodiesusedcanbefoundinSupplementalTable2.
GAPDHwasusedasaninternalcontrol.
CellviabilityassayCells(5*103)wereseededin96-wellplatesfor12h,followedbytreatmentwithcisplatin(Sigma,USA)atvariousconcentrations(1,5,10,20,50,100,500,1000M)andpaclitaxelatvariousconcentrations(0.
001,0.
01,0.
1,1,10,100m).
Afterincubationfor48JournalofCancer2020,Vol.
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Thepercentageofviablecells(%)=(absorbanceoftreatedsample/absorbanceofuntreatedsample)*100%.
ColonyformingassayCells(400cells/well)wereseededin6-wellplates.
After2weeksofculture,colonieswerefixedby4%paraformaldehydeandwerestainedwith0.
5%crystalviolet.
Thenumberofcolonies(≥100mindiameter)wascountedwithamicroscope.
SphereformingassayNCI-H1299cellsandNCI-H1975cellswereseededinultra-lowattachment6-wellplates(Corning,USA)andculturedinDMEM/F12supplementedwithB27,20ng/mlEGF,20ng/mlbFGF,and10ng/mlHGF.
CellswereincubatedinaCO2incubator2weekslater;sphereswerecountedundermicroscope(Olympus,Japan).
FlowcytometryCellsweretreatedwithdoxorubicin(0.
5g/ml)for48h.
Next,cellswereharvestedandstainedinbindingbuffer,AnnexinV-APCand7-AADasprovidedbytheAnnexinV-APC/7-AADApoptosisDetectionKit(KeyGENBioTECH,China).
Analysiswasdeterminedusingaflowcytometer.
StatisticalanalysisAllexperimentswereconductedatleastintriplicate.
Allthedatawerepresentedasthemeans±standarderrorofthemean(SEM).
StatisticalanalyseswereperformedusingStudent'st-testwithGraphPadPrism6(GraphPadSoftware,USA).
Inallcases,P50%)(Figure2B).
Therefore,wefirstobservedtheeffectsofZIC2onexpressionsofCSC-associatedmarkersincludingEpCAM,OCT4,SOX2,NANOG,C-MycandBmi-1.
ResultsshowedthatZIC2knockdownsignificantlyreducedmRNAexpressionoftheseCSC-associatedmarkerexpressioninNCI-H1299cells(Figure2C).
Consistently,WBassaysconfirmedthefindingsofRT-PCRassays(Figure2D).
Next,theeffectofZIC2expressiononsphere-formingcapacityofLACcellswasassessed.
WefoundthatdownregulationofZIC2greatlydecreasedthenumbersofspheroidcells(Figure2E).
Last,weexaminedeffectsofZIC2onCSCtraitsviaserialtumorigenesisexperiments.
ResultsshowedthatZIC2knockdownobviouslyinhibitedtumorformationatalldensitieswetested(Figure2F),confirmingthecriticalroleofZIC2inmaintainingCSCstraitsinNCI-H1299cells.
KnockdownofZIC2sensitizedLACcellstoconventionalchemotherapyregimensItiswellacknowledgedthatchemoresistanceactsasahallmarkofstemcell-liketrait[22],thus,wealsoinvestigatedtheinvolvementofZIC2indrugresistanceoflungadenocarcinomacells.
CCK8assayswereperformedtoevaluatetheinhibitoryratesofLACcellsunderdifferentdrugconcentrationtreatment.
WefoundthatZIC2knockdownresultedinamoresignificantreducedinhibitionrateswhentreatedwithcisplatininawiderangeofconcentrations(from5μMto500μm),comparedwiththecontrolcells(Figure3A).
Consistently,ZIC2knockdowncellsalsosensitizedNCI-H1299cellstopaclitaxeltreatment(Figure3B).
Tofurtherverifyabovefindings,wechosethetwosignificantinhibitoryconcentrationofcisplatin(10um,20um)andpaclitaxel(0.
01umand0.
1um)totreatLACcells,andcellproliferationpotentialsunderchemotherapyregimentreatmentwereassessedbycolonyformationassays.
WefoundthatZIC2knockdownsensitizedLACcellstoeithercisplatinorpaclitaxeltreatmentatbothtwoconcentrations,evidencedbydecreasednumberofclonesformed(Figure3CandD).
Finally,toconsolidateourpreviousresults,flowcytometryassayswereperformed.
Resultsshowedthat,comparedwithcontrolcells,ZIC2knockdownresultedinsignificantlyhigherapoptosisratesofLACcellswhentreatedwithcertainconcentrationofcisplatinorpaclitaxel(Figure3EandF).
Therefore,ourdatademonstratethatZICknockdownsignificantlysensitizedLACcellstoconventionalchemotherapyregimenttreatments.
OverexpressionofZIC2promotesstemcell-likephenotypeinLACToconfirmtheroleofZIC2inLACCSCmaintenanceandexpansion,wesuccessfullyconstructedthelentiviral-basedstableZIC2over-expressedNCI-H1975cells,confirmedbysignificantlyincreasedZIC2expressionaccordingtoRT-PCRandWBassays(Figure4A).
Afterwards,expressionsofCSC-associatedmarkers,sphere-forming,anddrugresistancecapacitieswerefurtherinvestigated.
ZIC2overexpressionresultedinincreasedexpressionlevelsofEpCAM,OCT4,SOX2,NANOG,C-MycandBmi-1accordingtoresultsofRT-PCRandWBassays(Figure4BandC).
Ofnote,theZIC2-OENCI-H1975cellsshowedanincreasedsphere-formingnumberswhencomparedwithcontrolcells(Figure4D).
Thus,ourdataindicatedthatforcedexpressionofZIC2inLACcellsdrovethesecellstowardsaCSC-likestate.
Finally,resultsfromserialtumorigenesisexperimentsshowedthatZIC2overexpressiongreatlyenhancedtumorformationcapacityofNCI-H1975cellsatalldensitieswetested(Figure2F).
JournalofCancer2020,Vol.
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ZIC2knockdownrestrictedCSC-associatedphenotypeinLACcells.
(A)ExpressionlevelsofZIC2innormalandLACcelllinesweredeterminedbyRT-PCRandWBassays.
(B)ZIC2knockdownefficiencieswereevaluatedbyRT-PCR(left)andWB(right)assays.
(C)EffectsofZIC2knockdownonCSC-associatedmarkerexpressioninNCI-H1299cellswereevaluatedbyRT-PCRassays.
(D)WBresultsfordescribingtheeffectsofZIC2knockdownonCSC-associatedmarkerexpressioninNCI-H1299cells.
(E)EffectsofZIC2knockdownonsphere-formingcapacityinNCI-H1299cells;Representativeimageswereshowninleftpanel.
(F)Ratiooftumor-freemiceafter12weeks'tumorformationafterinjectionofindicatednumbersofLACcells.
Imageswereshownintheleftpanel.
JournalofCancer2020,Vol.
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ZIC2knockdownsensitizedNCI-H1299cellstocisplatinandpaclitaxeltreatment.
(A)EffectsofZIC2knockdownontheresponsetodifferentconcentrationsofcisplatininNCI-H1299cellswereevaluatedbyCCK-8assays.
(B)EffectsofZIC2knockdownontheresponsetodifferentconcentrationsofpaclitaxelinNCI-H1299cellswereevaluatedbyCCK-8assays.
(C)ColonyformationassayswereconductedtoevaluatetheeffectsofZICknockdownoncisplatinresistanceinNCI-H1299cells;twoconcentrations(10and20mM)wereselectedaccordingtotheresultsofCCK-8assays.
(D)ColonyformationassayswereconductedtoevaluatetheeffectsofZICknockdownonpaclitaxelresistanceinNCI-H1299cells;twoconcentrations(0.
01and1mM)wereselectedaccordingtotheresultsofCCK-8assays.
(E)ApoptosisratesofZIC2-KDandcorrespondingparentalNCI-H1299cellsunderdifferentconcentrationsofcisplatintreatmentsweredeterminedbyFACS.
(F)ApoptosisratesofZIC2-KDandcorrespondingparentalNCI-H1299cellsunderdifferentconcentrationsofpaclitaxeltreatmentsweredeterminedbyFACS.
OverexpressionofZIC2rendersLACcellswithresistancetoconventionalchemotherapyregimensNext,wesetouttoinvestigatetheimpactsofforceexpressionofZIC2ondrugresistance.
CCK8assaysindicatedthatZIC2-overexpressedcellsexhibitedamoresignificanthigherresistancetovariousconcentrationsofcisplatintreatment(from5μMto500μm),comparedwithcontrolcells(Figure5A).
Similarly,ZIC2overexpressionalsosensitizedNCI-H1299cellstodifferentconcentrationsofpaclitaxeltreatment(Figure5B).
Colony-formationassaysindicatedthatZIC2overexpressionconferredconsiderablecisplatinandpaclitaxelresistancecapacitiestoLACcells,evidencedbyincreasednumberofclonesformedundersameregimentreatment(Figure5CandD).
Finally,FACSassaysindicatedthatcomparedwithcontrolcells,ZIC2overexpressionresultedinsignificantlylowerapoptosisratesofLACcellswhentreatedwithcertainconcentrationofcisplatinorpaclitaxel(Figure5EandF).
Takentogether,thesedatademonstratethatforcedZIC2expressionsignificantlysensitizedLACcellstoconventionalchemotherapyregimenttreatments.
ZIC2enhancesCSCtraitsviaanOCT4-dependentmannerAmongthenumerousstemness-relatedmoleculesassociatedwithZIC2expression,wefocusedonOCT4,whichshowedthemostsignificantmRNAexpressionchangesduetoZIC2alterations.
Also,OCT4wasreportedtoplaypivotalroleforCSCpropertiesinLAC[17].
ToinvestigateinteractionofZIC2withOCT4inLACCSCs,weoverexpressedOCT4inZIC2-KDNCI-H1299cells,whilesilencedOCT4expressioninZIC2-OENCI-H1975cells.
Asexpected,forcedexpressionofOCT4restoredexpressionofCSC-associatedmarkersinZIC2-KDNCI-H1299cells,whereasknockingdownOCT4greatlyabolishedthepromotionaleffectsofZIC2overexpressiononCSC-associatedmarkerexpressionJournalofCancer2020,Vol.
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Moreover,OCT4overexpressionsuccessfullyrescuedthesphere-formingcapacityinNCI-H1299cells,evidencedbyincreasednumberofspheroidsformed.
Onthecontrary,OCT4silencedramaticallyattenuatedthesphere-formingcapacityrenderedbyZIC2overexpressioninNCI-H1975cells(Figure6C).
Moreimportantly,upregulationofOCT4reversedtheZIC2knockdown-inducedcisplatinandpaclitaxelsensitizationinNCI-H1299cellsaccordingtoCCK-8assays(Figure6D),whereasdownregulationofOCT4significantlyabolishedZIC2-mediatedcisplatinandpaclitaxelresistanceinNCI-H1975cells(Figure6E).
InconcordancewiththeresultsofCCK-8assay,FACSresultsdemonstratedthattheapoptosisratesofZIC2-KDNCI-H1299cellsundercisplatinorpaclitaxeltreatmentsweresignificantlyreduceduponOCT4overexpression(Figure6F).
Contrarily,OCT4knockdownincreasedtheapoptosisratesinZIC2-OENCI-H1975cellsatbothtwoconcentrations(Figure6G).
Together,ourfindingsstronglyindicatedthatZIC2promotedCSCtraitsinLACviaup-regulatingOCT4expression.
Figure4.
ZIC2overexpressionpromotedCSC-associatedphenotypeinNCI-H1975cells.
(A)EfficienciesofZIC2overexpressioninNCI-H1975cellswerevalidatedbyRT-PCRandWBassays.
(B)EffectsofZIC2overexpressiononCSC-associatedmarkerexpressionweredeterminedbyRT-PCRassays.
(C)EffectsofZIC2overexpressiononCSC-associatedmarkerexpressionweredeterminedbyWBassays.
(D)EffectsofZIC2overexpressiononsphere-formingcapacityinNCI-H1975cellswereevaluated;Representativeimageswereshownasleftpanel.
(F)Ratiooftumor-freemiceafter12weeks'tumorformationafterinjectionofindicatednumbersofLACcells.
Imageswereshownintheleftpanel.
JournalofCancer2020,Vol.
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ZIC2overexpressioninducedresistancetocisplatinandpaclitaxelinNCI-H1975cells.
(A)EffectsofZIC2overexpressionontheresponsetodifferentconcentrationsofcisplatininNCI-H1975cellswereevaluatedbyCCK-8assays.
(B)EffectsofZIC2overexpressionontheresponsetodifferentconcentrationsofpaclitaxelinNCI-H1975cellswereevaluatedbyCCK-8assays.
(C)ColonyformationassayswereconductedtoevaluatetheeffectsofZICoverexpressiononcisplatinresistanceinNCI-H1975cells;twoconcentrations(10and20mM)wereselectedaccordingtotheresultsofCCK-8assays.
(D)ColonyformationassayswereconductedtoevaluatetheeffectsofZICoverexpressiononpaclitaxelresistanceinNCI-H1975cells;twoconcentrations(10and20mM)wereselectedaccordingtotheresultsofCCK-8assays.
(E)ApoptosisratesofZIC2-OEandcorrespondingparentalNCI-H1299cellsunderdifferentconcentrationsofcisplatintreatmentsweredeterminedbyFACS.
(F)ApoptosisratesofZIC2-OEandcorrespondingparentalNCI-H1299cellsunderdifferentconcentrationsofpaclitaxeltreatmentsweredeterminedbyFACS.
DiscussionZIC2isageneoriginallyidentifiedbytheirhomologytothedrosophilaodd-pairedgenes.
HeterozygousdeletionsandmutationsofZIC2cancauseseverebrainmalformation[23,24].
Thus,itiscriticalforthedevelopmentofCNS[25].
RecentreportshaveshownacorrelationbetweenZIC2expressionandcarcinogenesis.
ZIC2isaberrantlyactivatedinvariouscancertypes,suchashepatocellularcarcinoma[19],bladdercancer[26],andcervicalcancer[27].
ZIC2isupregulatedinthesetumorsandisassociatedwithahigherstageoftumorsandpoorprognosis.
Therefore,ZIC2isregardedasanoncogene.
However,therearenodataregardingtheexpressionofZIC2inLACanditsclinicalsignificance.
Inourstudy,werevealedthatZIC2washighlyexpressedinLACtumortissuesandindicatedapooroverallsurvivalofLACpatients,whichsuggestedthatZIC2maybeapotentialprognosticbiomarkerforLACpatients.
CSCshavebeenidentifiedinmanysolidtumorsandplayacentralroleintumorinitiation,progression,andtherapeuticresistance[28].
AbetterunderstandingofthemolecularmechanismsessentialforCSCmaintenanceandexpansionwouldshedlightontheenhancementofclinicalmanagement.
Importantly,specificallyandeffectivelytargetingCSCsiswidelyconsideredasapotentialstrategytoimprovetheoverallsurvivalratesofcancerpatients[19,29].
PreviousstudiesrevealedthatZIC2wasrequiredfortheself-renewalmaintenanceofliverCSCs[19].
Additionally,arecentreportshowedthatZIC2ishighlyexpressedincervicalcancerandassociatedwithactivationofHedgehogsignaling,acrucialcancerstemnesspathway[27].
However,theLACCSCbiologyremainslargelyunknownandwhetherZIC2playaroleinLACCSChasnotbeenreportedpreviously.
Here,usingbothloss-of-functionandgain-of-functionexperiments,weprovideevidencefortheroleofZIC2intheregulationofLACcellself-renewalandstemness.
WefoundZIC2expressionwaspositivelyassociatedwiththeexpressionofstemmed-relatedmoleculesandwasessentialforthespheroidcellformation.
JournalofCancer2020,Vol.
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org6078Figure6.
ZIC2promotedCSCtraitsinLACvieup-regulatingOCT4expression.
(A)OCT4expressionwasrestoredinZIC2-KDNCI-H1299cells(upper),orsilencedinZIC2-OENCI-H1975cells(lower),andtheirimpactsonCSC-associatedmarkerexpressionwereevaluatedbyRT-CPR.
(B)OCT4expressionwasrestoredinZIC2-KDNCI-H1299cells(left),orsilencedinZIC2-OENCI-H1975cells(right),andtheirimpactsonCSC-associatedmarkerexpressionwereevaluatedbyWBassays.
(C)OCT4expressionwasrestoredinZIC2-KDNCI-H1299cells(upper),orsilencedinZIC2-OENCI-H1975cells(lower),andtheirimpactsonsphere-formingcapacitieswereevaluated.
(D)OCT4expressionwasrestoredinZIC2-KDNCI-H1299cells,anditsimpactsontheresponsetodifferentconcentrationsofcisplatin(left)orpaclitaxel(right)weredetectedbyCCK-8assays.
(E)OCT4expressionwassilencedinZIC2-OENCI-H1975cells,anditsimpactsontheresponsetodifferentconcentrationsofcisplatin(left)orpaclitaxel(right)weredetectedbyCCK-8assays.
(F)OCT4expressionwasrestoredinZIC2-KDNCI-H1299cells,anditsimpactonapoptosisrateunderdifferentconcentrationsofcisplatin(left)orpaclitaxel(right)treatmentwasdeterminedbyFACS.
(G)OCT4expressionwassilencedinZIC2-OENCI-H1975cells,anditsimpactonapoptosisrateunderdifferentconcentrationsofcisplatin(left)orpaclitaxel(right)treatmentwasdeterminedbyFACS.
Thedevelopmentofchemoresistanceisamajorobstaclefortheeffectivetreatmentofhumanmalignancies[30].
Cisplatinandpaclitaxelarefist-linechemotherapeuticagentsusedforvariouscancersJournalofCancer2020,Vol.
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However,theresponsetothesedrugsforLACpatientssoondisappears,leadingtopatientdeathandthepoorfive-yearsurvivalrates[32].
Chemoresistanceisacomplexphenomenonandrevealingthemolecularmechanismsrelatedtoinherentandacquiredresistanceisimportanttoovercomedrugresistance.
ConsideringthatcancerstemcellsareresponsibleforthetumorchemoresistanceandthefunctionalrolesofZIC2inCSCs,theinfluenceofZIC2oncisplatinandpaclitaxelresistanceneededtobedeeplyinvestigatedinlungadenocarcinomacells.
OurstudyfoundthatZIC2overexpressioncellsdramaticallyenhanceddrugresistancepropertiesinLACcells.
Therefore,ourstudyidentifiedanoveltherapeutictargetforreversingdrugresistanceinLAC,anddevelopingstrategytolowerZIC2expressionmayincreasetheefficacyofcisplatinandpaclitaxeltreatmentinLACtumor.
OCT4wasidentifiedasastemcelltranscriptionfactorandhadbeenreportedtobehighlyexpressedinLACtumor[12,17].
OCT4actsasagatekeeperinthebeginningofmammaliandevelopmentandregulatesthetranscriptionofmanygenes,suchasSOX2,NANOGandc-MYC[12].
Indeed,OCT4wasthekeygenebetweentumorinitiationandprogressionviathedirectmediationofkeymoleculesthatregulatecancerstemnesstraits[33].
Moreover,highOCT4expressionwasfoundtobecloselyassociatedwithpooroutcomesinLACpatients[17].
ZIC2,astranscriptionfactor(TF),behavedastheothercorepluripotentTFs(SOX2,NANOG,c-MYC)incancerstemcells[24].
Interestingly,theassociationbetweenZIC2andOCT4isreportedinlivercancer[19].
Inaddition,ZIC2isrequiredfortheexpressionofOCT4.
Notsurprisingly,lungcancersubtypesmayalsosharecoreregulatorygenesandcommonsignalingpathways.
OurinvitroassaysfurtherdemonstratedthatZIC2actedasacriticaloncogeneresponsibleforself-renewalandstemnessofLACbyupregulationofOCT4.
Therefore,ZIC2-inducedOCT4maybeconsideredasamasterregulatortomaintainandgovernLACCSCs.
ThissuggeststhattheregulatorypathwaybetweenZIC2andOCT4maybesignificantforthestabilizationofstemness-likestate.
Insummary,ourstudyprovidedvariousevidencetolinkhighexpressionofZIC2withtheacquisitionofLACCSC-likeproperties,andproposedanovelmechanismofLACCSC-liketraitsmediatedbyZIC2.
WeunveiledthatZIC2maintainedtheself-renewal,stemnessandchemoresistancecapacitiesinanOCT4-dependentmanner.
Toourknowledge,thisisthefirststudytorevealthatZIC2exerteddramaticeffectsonCSC-likepropertiesinhumanLACtumor.
TheZIC2-OCT4networkwillfacilitatetheevaluationofthepotentialtherapeuticefficacyofchemotherapyandpredictpatientsensitivitytotreatment.
SupplementaryMaterialSupplementaryfiguresandtables.
http://www.
jcancer.
org/v11p6070s1.
pdfAcknowledgmentsDataavailabilityThedatasetsusedand/oranalyzedduringthecurrentstudyareavailablefromthecorrespondingauthoronreasonablerequest.
EthicsapprovalandconsenttoparticipateThepresentstudywasapprovedbytheMinhangHospitalResearchEthicsCommittee,andallindividualsprovidedtheirinformedconsent.
Humanecareofanimalswasobjectedtothe"GuidefortheCareandUseofLaboratoryAnimals"criteriaofNationalAcademyofScience(NationalInstituteofHealthpublication86-23,revised1985).
Authors'contributionStudydesign:DWJ,andQC;Acquisition,analysis,andinterpretationofthedata:WHW,NZ,QC,andDWJ;Draftingofthemanuscript:WHW,NZ,QC,andDWJ;Criticalrevisionofthemanuscript:WHW,NZ,QC,andDWJ;Approveofthefinalmanuscript:allauthors.
CompetingInterestsTheauthorshavedeclaredthatnocompetinginterestexists.
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