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Increased expression of the dsRNA-Activated Protein kinase PKR in breast cancer promotes sensitivity to doxorubicin
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Title: Increased expression of the dsRNA-Activated Protein kinase PKR in breast cancer promotes sensitivity to doxorubicin
Series Title: PLoS ONE 7(9): e46040. doi:10.1371/journal.pone.0046040
Physical Description: Journal Article
Creator: Bennett, Richard
Publisher: PLoS One
Place of Publication: PLoS One
Publication Date: 9-24-12
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Abstract: It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition, the rate of eIF2a phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-a, which increases PKR expression, or salubrinal, which increases eIF2a phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2a phosphorylation may be beneficial for the treatment of breast cancer.
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Publication Status: Published
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IncreasedExpressionofthedsRNA-ActivatedProtein KinasePKRinBreastCancerPromotesSensitivityto DoxorubicinRichardL.Bennett,AubreyL.Carruthers,TengHui,KrystalR.Kerney,XiangfeiLiu,W.StratfordMay,Jr. *DepartmentofMedicine,DivisionofHematologyandOncologyandtheUniversityofFloridaShandsCancerCenter,UniversityofFlorida,Gainesville ,Florida,UnitedStates ofAmericaAbstractIthasbeenreportedthattheexpressionandactivityoftheinterferon-inducible,dsRNA-dependentproteinkinase,PKR,is increasedinmammarycarcinomacelllinesandprimarytumorsamples.ToextendthesefindingsanddeterminehowPKR signalingmayaffectbreastcancercellsensitivitytochemotherapy,wemeasuredPKRexpressionbyimmunohistochemical stainingof538casesofprimarybreastcancerandnormaltissues.Significantly,PKRexpressionwaselevatedinductal, lobularandsquamouscellcarcinomasorlymphnodemetastasesbutnotineitherbenigntumorspecimensorcasesof inflammationcomparedtonormaltissues.Furthermore,PKRexpressionwasincreasedinprecancerousstagesofmammary cellhyperplasiaanddysplasiacomparedtonormaltissues,indicatingthatPKRexpressionmaybeupregulatedbythe processoftumorigenesis.TotestthefunctionofPKRinbreastcancer,wegeneratedMCF7,T-47DandMDA-MB-231breast cancercelllineswithsignificantlyreducedPKRexpressionbysiRNAknockdown.Importantly,whileknockdownofPKR expressionhadnoeffectoncellproliferationundernormalgrowthconditions,MCF7,T-47DorMDA-MB-231cellswith reducedPKRexpressionortreatedwithasmallmoleculePKRinhibitorweresignificantlylesssensitivetodoxorubicinor H2O2-inducedtoxicitycomparedtocontrolcells.Inaddition,therateofeIF2 a phosphorylationfollowingtreatmentwith doxorubicinwasdelayedinbreastcancercelllineswithdecreasedPKRexpression.Significantly,treatmentofbreastcancer lineswithreducedPKRexpressionwitheitherinterferona ,whichincreasesPKRexpression,orsalubrinal,whichincreases eIF2 a phosphorylation,restoreddoxorubicinsensitivitytonormallevels.Takentogethertheseresultsindicatethat increasedPKRexpressioninprimarybreastcancertissuesmayserveasabiomarkerforresponsetodoxorubicin-containing chemotherapyandthatfuturetherapeuticapproachestopromotePKRexpression/activationandeIF2 a phosphorylation maybebeneficialforthetreatmentofbreastcancer.Citation: BennettRL,CarruthersAL,HuiT,KerneyKR,LiuX,etal.(2012)IncreasedExpressionofthedsRNA-ActivatedProteinKinasePKRinBreastCancer PromotesSensitivitytoDoxorubicin.PLoSONE7(9):e46040.doi:10.1371/journal.pone.0046040 Editor: ZhaozhongHan,VanderbiltUniversity,UnitedStatesofAmerica Received May22,2012; Accepted August28,2012; Published September24,2012 Copyright: 2012Bennettetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwasfundedbygrantNIH/NHLBIR01-HL054083.Thefunderhadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,or preparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:smay@ufl.eduIntroductionTheinterferon(IFN)-inducible,double-strandedRNA-activated proteinkinase,PKR,ispresentinmostmammaliancellsinalatent orinactivestate.Ithasbeenwellstudiedasanimportant componentoftheIFN-stimulatedhostantiviraldefensemechanism.Inthiscontext,PKRisinducedbyIFNandactivatedby viraldouble-strandedRNAtocatalyzephosphorylationofeIF2 a resultinginglobalproteinsynthesisinhibitionandinitiationof apoptosis.Significantly,ourlaboratoryandothershavedeterminedthatPKRmaybeactivatedbyavarietyofcellular stressessuchashematopoieticgrowthfactorstarvation,inflammatorycytokinesandchemotherapyagenttreatment.[1,2,3]In additiontoaninhibitoroftranslation,PKRhasbeenreportedto haveanimportantroleinsignalingpathwayssuchasNFk B,p53 andSTAT1thatregulateproliferationandapoptosisduring cellularstress.[4,5,6,7,8,9,10,11,12,13,14,15]Thus,PKRmay serveasaguardianofthecellthatfacilitatestheresponseto diversestressstimuli. TheroleofPKRintumorigenesisisnotwellcharacterized. Ingeneral,PKRisconsideredtohaveatumorsuppressor functionsinceincreasedPKRactivityhasbeencorrelatedwith decreasedcellproliferationandananti-tumoractivity [16,17,18].Insupportofthis,mutantformsofPKRand PKR’sdownstreamtarget,eIF2 a ,aswellasinhibitorsofPKR suchasTRBPorp58caninducetransformationofcells. [19,20]Furthermore,thelossofPKRcatalyticactivityhasbeen observedinB-cellchroniclymphocyticleukemiapatient samples,andaninactivatingpointmutantinPKR’sdsRNA bindinghasbeendetectedinasmallsetofpatientswithacute lymphoblasticleukemiaofT-celllineage.[21,22]ThePKR geneislocatedon2p21-22,achromosomalregionthathas beenassociatedwithlargecelllymphoma,myelodysplastic syndromeandacutemyelogenousleukemia.[23,24,25,26,27] Inaddition,thePKRgeneistranscriptionallyregulatedbyIFNs a and c viaIRF-1,anddownregulationofPKRhasbeen showntooccurin5q-associatedleukemiasthatdeletetheIRF1gene.[28,29,30,31]Significantly,ithasbeenrecentlyreported thatprimarynon-smallcelllungcancer(NSCLC)sampleshave PLOSONE|www.plosone.org1September2012|Volume7|Issue9|e46040

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decreasedPKRexpressioncomparedtonormalbronchial epithelium.[32]Furthermore,lossofPKRexpressioncorrelates withamoreaggressivebehaviorwhilehighPKRexpression predictsasubgroupofNSCLCpatientswithafavorable outcome.[32]Collectively,thesefindingssuggestthatPKRmay playanimportantroleintumorsuppressionandthatinhibition ofPKRactivityisassociatedwithtumorigenesis. Asaninitiatorofapoptosisinresponsetocellularstress,PKR maymediatethesensitivityofcancercellstochemotherapy.For example,PKRisactivatedbytheanthracyclinedoxorubicin (DOX),acommonlyusedtreatmentforawiderangeof cancers.[33]FollowingDOXapplication,PKRhasbeen reportedtoinduceapoptosisofcancercelllinesbymechanisms dependentoneIF2 a phosphorylation,p53phosphorylationand JNKactivation.[33,34]Importantly,inamousexenograft model,coloncancercellswithreducedPKRexpressionmore rapidlyestablishedsolidtumorsthatwereresistanttoDOXor etoposidetreatmentcomparedtocontrolcells.[35]Inaddition, ithasbeenreportedthatPKRpromotes5-Fluorouracil(5-FU)inducedapoptosisbyamechanismdependentoneIF2 a phosphorylation.[36]Significantly,knockdownofPKRexpressionincolonandbreastcancercelllinesresultedinadecreased sensitivityto5-FUandeliminatedtheabilityofIFN a to improve5-FUcytotoxicity.[36]. TobetterunderstandtheroleofPKRsignalinginbreast cancercellproliferationandresponsetochemotherapy,we analyzedPKRexpressionandfunctioninbothprimarybreast cancertissuesand3commonbreastcancercelllines.Previous workfromotherlaboratoriesindicatesPKRexpressionis elevatedbothinprimaryductalcarcinomatissuescompared tonormalluminalductalepithelialsamples,andinbreast cancerderivedcelllinesthannontransformedmammary epithelialcelllines.[37,38,39].Toextendthesefindings,we measuredPKRexpressionbyimmunohistochemical(IHC) stainingofprimarybreastcancertissuemicroarrayscontaining 538cases.Significantly,resultsindicatethatPKRiselevatedin invasiveductal,lobularandsquamouscellcarcinomasaswellas inregionallymphnodemetastasiscomparedtonormalbreast tissue.Furthermore,PKRexpressionisincreasedinprecancerousstagesofmammarycellhyperplasiaanddysplasiacompared tonormaltissuesbutnotcasesofbreasttissueinflammation, indicatingthatPKRexpressionmaybeupregulatedduring tumorigenesis.Inaddition,weinvestigatedtheresponseto DOXinbreastcancercelllinesMCF7,T-47DorMDA-MB231withsignificantlyreducedPKRexpressionbysiRNA knockdown.Importantly,breastcancercelllineswithreduced PKRexpressionortreatedwithaPKRinhibitorareless sensitivetoDOXorH2O2-mediatedcytotoxicitycomparedto controlcells.Furthermore,followingtreatmentwithDOX, breastcancercelllineswithreducedPKRexpressionhave adecreasedrateofeIF2 a phosphorylationcomparedtocontrol cells.Inaddition,treatmentofMCF7,T-47DorMDA-MB-231 cellswithIFN a ,toincreasePKRexpression,orwithsalubrinal, toincreasephosphorylatedeIF2 a ,increasesDOXcytotoxicity andrestoresDOXsensitivityincellswithreducedPKR expressiontothatofcontrolcells.Takentogethertheseresults suggestthatincreasedactivationofPKR-eIF2 a signaling observedinbreastcancerspecimensmaycontributetothe therapeuticindexofDOX-containingchemotherapy.Thus, PKRexpressionmayserveasabiomarkerforDOXsensitivity andstrategiestoincreasePKR-eIF2 a signalingmaybe therapeuticallyusefulforbreastcancerinthefuture.Results PKRExpressionisIncreasedinPrimaryBreastCancer TissuesToinvestigatetheclinicalrelevanceofPKRexpressioninbreast cancer,wemeasuredPKRlevelbyhighthroughputimmunohistochemical(IHC)analysisoftissuemicroarrayscontaining538 primarysamples.Thearraysconsistedof154normalorcancer adjacentnormal,243malignant,47lymphnodemetastasis,37 benignfibroadenoma,31hyperplasia,10dysplasia,and16 inflammationcases.Malignantcasesconsistedof167invasive ductalcarcinomas,34invasivelobularcarcinomas,2carcinosarcomas,8cystosarcomaphyllodes,4lobularcarcinomainsitu,9 medullarycarcinomas,8mucinousadenocarcinomas,3mucous carcinomas,4Paget’sdisease,and4squamouscellcarcinomas. IHCstainingforPKRwasscoredonascaleof0(nostaining)to9 (strong,100%staining).Thenumberoftissuecoresexaminedper casevariedfrom1to3,andPKRstainingscoresforcaseswith duplicateortriplicatecoreswereaveraged. Significantly,IHCstainingforPKRwasincreasedinmalignant comparedtonormalmammaryepithelialtissue(meanscore6.892 vs.4.569,P 0.0001;Figure1andTable1).Furthermore, increasedPKRexpressioncomparedtonormaltissueswas statisticallysignificantforthemoreaggressivetumorsincluding invasiveductalandlobularcarcinomasaswellassquamouscell carcinomas(Figure1andTable1).Allgrades(1to3)ofinvasive ductalcarcinomadisplayeduniformlyelevatedPKRexpression comparedtonormaltissues.However,nosignificantdifference betweenthetumorgrades(1to3)ofinvasiveductalcarcinomas wasobserved(Table1).Moreover,PKRwasincreasedinlymph nodemetastasiscomparedtonormaltissues(6.887vs.4.569, P 0.0001;Table1).Incontrast,nosignificantdifferenceinPKR levelscouldbeobservedbetweenothertypesofbreastcancer examinedorbetweenbenignvs.normalspecimens(Table1). Inaddition,toassessthepointduringmalignanttransformation thatPKRexpressionmaybeincreased,weanalyzedprecancerous andinflammationtissuespecimens.Nosignificantdifferencein PKRexpressionwasobservedinbreastinflammation(including casesofmastitisorchronicinflammation)comparedtonormal specimens(Table1andFigure2A).Incontrast,potentially precanceroustissuesincludinghyperplasiasanddysplasiasdemonstratedsignificantlyelevatedPKRexpressioncomparedto normal(Table1andFigure2B).TheseresultssuggestthatPKR expressionmaybeincreasedduringtheprocessofmalignant transformationbyanunknownmechanism.PKRExpressioninBreastCancerCellLinesisRequiredfor CellInvasionTodeterminethesignificanceofincreasedPKRexpressionon breastcancercellproliferationandsensitivitytochemotherapy agents,wetestedtheroleofPKRinthebreastcancercelllines MCF7,T-47DandMDA-MB-231.Interestingly,basalPKR phosphorylationatthreonine451,acriticalsiteofPKRautophosphorylation,wasdramaticallyhigherinbreastcancercelllines MCF7,T-47DandMDA-MB-231comparedtothe‘‘nontransformed’’mammaryepithelialcelllineMCF10A(Figure3A).To determinewhetherPKRlevelandactivitymayberequiredfor proliferationortumorresponsetochemotherapyagents,we employedasiRNAstrategytoknockdownPKRby 90%in breastcelllinesMCF7andT-47Dand 50%inMDA-MB-231 (Figure3B).Significantly,breastcancercelllineswithreduced PKRexpressiondidnotdisplayadefectincellproliferationunder standardgrowthconditions(Figure3C–E).RoleofPKRinBreastCancer PLOSONE|www.plosone.org2September2012|Volume7|Issue9|e46040

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RoleofPKRinBreastCancer PLOSONE|www.plosone.org3September2012|Volume7|Issue9|e46040

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Sinceourlaboratoryandothershavepreviouslyfoundthat PKRisrequiredfortheresponsetoserumwithdrawaland growthfactorstarvation,wetestedwhetherPKRexpression effectedbreastcancercellinvasion.[1,3]Briefly,cellswere seededintoaBoydenchamberinserum-freemediumwhile serum-containingmediumwasplacedinthelowerchamber. Cellinvasionthroughtheextracellularmatrixwasscoredafter 24hours.Significantly,MCF7,T-47DandMDA-MB-231cells withreducedPKRexpressiondisplayedreducedcellinvasion (Figure3F-H).TheseresultsmayindicatethatthePKRdependentresponsetogrowthfactorstarvationisrequiredfor cellinvasionandthatincreasedPKRexpressioninbreast cancercellsmaypromotecellinvasion.BreastCancerCellswithReducedPKRExpressionareLess SensitivetoDoxorubicinTodeterminetheroleofPKRonbreastcancercellresponseto chemotherapyagents,MCF7,T-47DandMDA-MB-231cells expressingeithersiRNAtoPKRorcontrolsiRNAweretreated withdoxorubicin(DOX)andviabilitywasmeasuredbyTrypan bluedyeexclusionassay.Importantly,breastcancercelllineswith reducedPKRexpressionaresignificantlylesssensitiveto increasingconcentrationsofDOXthancontrolcells(Figure4A– C).Significantly,following48hourstreatmentwith10 m MDOX, MCF7orT-47DcellswithreducedPKRdisplayanalmost25% increaseinsurvivalcomparedtocellsexpressingacontrolsiRNA (Figure4AandC).Similarly,MDA-MB-231cellswithreduced PKRdisplayanalmost15%increaseincellsurvivalcomparedto controlcells(Figure4E).Inaddition,followingtreatmentofMCF7 cellswith5 m MDOXforvarioustimes,cellswithreducedPKR displayareducedrateofcelldeathcomparedtocontrolcells (FigureS1A).Inaddition,toconfirmthatDOX-treatedbreast cancercelllinesdiebyapoptosis,weperformedaTUNELassay. Importantly,DOXtreatmentinducesDNAfragmentation detectedbyTUNELassayinbreastcancercelllinesandfollowing 24hourstreatmentwith5 m Mdoxorubicin,MCF7andT-47D cellswithreducedPKRexhibitareductioninTUNELpositive cellscomparedtocellsexpressingacontrolsiRNA(FigureS1B). TakentogethertheseresultsindicatethatPKRexpression promotessensitivitytoDOX-inducedapoptosisinbreastcancer cells. SinceoneconsequenceofDOXtreatmentisincreasedROS thatmaymediatecytotoxicity,wetestedthesensitivityofbreast cancercelllineswithreducedPKRtoH2O2treatment. Significantly,following48hourstreatmentwithincreasing concentrationsofH2O2,MCF7,T-47DandMDA-MB-231cells expressingPKRsiRNAhaveareducedrateofcelldeath comparedtocontrolsiRNAcells(Figure4B,D,andF). Importantly,following48hourstreatmentwith2mMH2O2, MCF7cellswithreducedPKRexpressiondisplayan 25% increaseinviabilitycomparedtocontrolcells(Figure4B). Furthermore,following48hourstreatmentwith4mMH2O2, T-47DorMDA-MB-231cellswithreducedPKRexpression displayan 20%increaseinviabilitycomparedtocontrolcells (Figure4Dand4F). Inaddition,wetestedwhetherPKRlevelmayeffectbreast cancercelllinesensitivitytoanotherstandardandpotent chemotherapyforbreastcancer,paclitaxel.Interestingly,MCF7Figure1.PKRissignificantlyelevatedinprimarybreastcancertissuescomparedtonormalorbenigntissues. Representativeresults fromIHCstaining(20 6 magnificationshown).PKRisstainedbrownandnucleiarestainedblue.Arrowshighlightpositivelystainedcells. A .Normal breasttissues.Leftpanel:44yearoldfemale;Rightpanel:35yearoldfemale. B .Benign:Leftpanel:44yearoldfemale,adenosis;Right:35yearold female,bluntductadenosis. C .Invasiveductalcarcinomas:Leftpanel:57yearoldfemale,IDCnototherwisespecified,Stage3;RightPanel:45year oldfemale,grade1T4N1M0. D .Invasivelobularcarcinomas:Leftpanel:35yearoldfemale;Rightpanel:38yearoldfemale. doi:10.1371/journal.pone.0046040.g001 Table1. ImmunohistochemicalanalysisofPKRexpressioninnormalandbreastcancerTMAspecimens.PathologydiagnosisNo.ofcasesMeanPKRscorePNormal 1544.569 6 0.2465reference Normal 933.675 6 0.2700 Canceradjacentnormal615.932 6 0.4119 Malignant 2436.892 6 0.1684 0.0001 Ductalcarcinomas 1677.283 6 0.1854 0.0001 Grade1 197.474 6 0.6247 0.0001 Grade2 877.339 6 0.2551 0.0001 Grade3 268.244 6 0.3477 0.0001 Lobularcarcinomas 347.735 6 0.2804 0.0001 Squamouscellcarcinoma48.250 6 0.75000.0179 Others:Carcinosarcoma(N=2),Cystosarcomaphyllodes(N=8),LobularcarcinomaInSitu (N=4),Medullarycarcinoma(N=9),Mucinousadenocarcinoma(N=8),Mucouscarcinoma (N=3),Paget’sdisease(N=4) 384.325 6 0.47890.6576 Lymphnodemetastasis 476.887 6 0.3530 0.0001 Benign 374.878 6 0.41860.5697 Hyperplasia 316.613 6 0.36610.0005 Dysplasia 108.200 6 0.52810.0003 Inflammation 164.146 6 0.48680.5892 doi:10.1371/journal.pone.0046040.t001 RoleofPKRinBreastCancer PLOSONE|www.plosone.org4September2012|Volume7|Issue9|e46040

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cellswithreducedlevelsofPKRdisplayapproximatelythesame sensitivitytopaclitaxelascontrolcells(FigureS1C).Furthermore, co-treatmentofMCF7cellswiththecombinationofDOXand paclitaxel,demonstratesthatPKRhasnoeffectonthesynergy betweenthesetwocompounds,sinceanydifferenceinviabilitycan beattributedtothereducedsensitivitytoDOXpreviously observed(FigureS1D).Thus,PKRmaybeimportantforthe responsetoDOXandROSbutnotinvolvedintheresponseto microtubulestress.PKRActivityandIncreasedExpressionPromote DoxorubicinSensitivitySincehighlevelsofphospho-threonine451PKRarepresentin breastcancercelllinesthatmayindicatehighlevelsofbasalPKR activity,wetestedwhetherspecificinhibitionofPKRactivity couldprotectcellsfromDOX-inducedcytotoxicity.Briefly, MCF7,T-47DorMBA-MD-231cellsweretreatedeitherwith 2.5 m MDOXaloneorwith2.5 m MDOXandincreasing concentrationsofasmallmoleculePKRinhibitor(PKRI)for48 hours.Importantly,treatmentwithincreasingconcentrationsof PKRIinhibitedsensitivitytoDOXforthethreebreastcancercell linestested.Forinstance,co-treatmentwith2.5 m MDOXand 1 m MPKRIresultedina 20%reductionincytotoxicity comparedtotreatmentwith2.5 m MDOXaloneinallthreecell lines(Figure5A).TheseresultsindicatethatPKRactivitymay facilitatefullandpotentsensitivitytodoxorubicin. Since,PKRisawell-characterizedIFN-induciblegene,wenext testedwhetherincreasingthelevelofPKRbyIFNtreatmentcould affectsensitivitytoDOX.Treatmentofbreastcancercelllines withIFN a significantlyincreasedthelevelofPKRasdetectedby westernblotting(Figure5B).Next,breastcancercelllineswerecotreatedwithboth500Units/mlofIFN a andincreasingDOX concentrationsfor48hours.Importantly,co-treatmentwithIFN a promoteda2–5foldincreaseinsensitivityofbreastcancercell linestoDOX(Figures5C–EcomparedtoFigures4A,CandE). Furthermore,DOXsensitivityofcelllinesexpressingPKRsiRNA wasnearlyrestoredtothelevelofcontrolcellsbyco-treatment withIFN a (Figure5C–E).Importantly,theabilityofIFN a to promotesensitivitytoDOXwasinhibitedbyco-treatmentwith PKRI(Figure5F).Theseresultsillustratethatincreasedexpression andactivityofPKRmaybecriticalforbreastcancercellsensitivity todoxorubicin.PhosphorylationofthePKRTarget,eIF2 a ,Promotes SensitivitytoDoxorubicinOnewell-definedmechanismbywhichPKRcanpromote apoptosisisbyphosphorylationofeIF2 a resultingininhibitionof proteinsynthesis.Therefore,weexaminedtherateofeIF2 a phosphorylationinbreastcancercelllinesfollowingtreatment withDOX.Significantly,eIF2 a phosphorylationisinducedin MCF7,T-47DandMDA-MB-231cellsfollowing4to8hours treatmentwith2.5 m MDOX(Figure6A,BandC;siControl cells).Incontrast,breastcancercelllineswithreducedPKR expressionbysiRNAknockdownhaveadelayedrateofeIF2 a phosphorylationfollowingDOXtreatmentwithsignificanteIF2 a phosphorylationnotobserveduntil24hoursof2.5 m MDOX treatment(Figure6A,BandC;siPKRcells).Asanindicatorof apoptosis,westernblottingwasalsoperformedforcleavedPARP followingtimesofDOXtreatment.Importantly,asignificantlevel ofcleavedPARPwasobservedinMCF7andMDA-MB-231 siRNAcontrolcellsfollowing16hoursofDOXtreatment (Figure6AandB;siControlcells).Incontrast,cleavedPARP waseithernotobservedorgreatlyreducedinMCF7andMDAMB-231cellswithreducedPKRexpressionbysiRNAknockdown (Figure6AandB;siPKRcells).Inaddition,cleavedPARPwasnot observedineitherT-47DcontrolorPKRsiRNAcellsunderthese conditions(datanotshown).Takentogethertheseresultssuggest thatphosphorylationofeIF2 a andconsequentapoptosisisdelayed incellswithreducedPKRcomparedtocontrolcellsfollowing DOXtreatment. TofurtherinvestigatewhetherinductionofeIF2 a phosphorylationmaypromotebreastcancercellsensitivitytoDOX,we treatedMCF7,T-47DandMDA-MB-231cellseitherexpressing controlorPKRsiRNAwithsalubrinal,aspecificinhibitorof eIF2 a phosphatasethathasbeenreportedtocauseanincreasein thelevelofphosphorylatedeIF2 a [40].Briefly,MCF7,T-47Dor MBA-MD-231cellswereeithertreatedwith2.5 m MDOXalone orco-treatedwith2.5 m MDOXandincreasingconcentrationsof salubrinalfor48hours.Importantly,treatmentwith20 m M salubrinalfor48hourssignificantlyincreasedthelevelof phosphorylatedeIF2 a inallcelllinestested(Figure7A).Furthermore,co-treatmentwithsalubrinalincreasedDOXcytotoxicityin bothcontrolsiRNAandPKRsiRNAexpressingcelllines (Figure7B–D).Significantly,salubrinaltreatmentofcellswith reducedPKRrestoredDOXsensitivitytothelevelofcontrolcells (Figure7B–D).Importantly,salubrinaltreatmentalonedidnot promotecelldeath(datanotshown).Theseresultsindicatethat phosphorylationofPKR’sdownstreamtarget,eIF2 a ,isimportant forthefullandpotentcytotoxiceffectofDOX.Furthermore, treatmentwithsalubrinalmaybeusedtorestoreDOXsensitivity tocellswithreducedPKRexpression.DiscussionWereportthatPKRexpressionissignificantlyupregulatedin primarybreastcancercomparedtonormalorbenignbreast epithelialtissue.Inaddition,PKRexpressionisincreasedin precancerousstagesofmammarycellhyperplasiaanddysplasia comparedtonormaltissuesbutnotcasesofbreasttissue inflammation.Thus,theoncogenictransformationprocessitself mayplayaroleinelevatedPKRexpressionsimilartowhathas beenobservedincoloncancer.[41]Furthermore,andpotentially oftherapeuticsignificance,resultsdemonstratethatelevatedPKR inbreastcancercelllinesmayfunctiontospecificallyenhance doxorubicin(DOX)-inducedapoptosisbyamechanismdependent oneIF2 a phosphorylation.Significantly,treatmentofbreast cancercelllineswithsalubrinal,topromoteeIF2 a phosphorylaFigure2.IncreasedPKRexpressioninbreasttissuespecimens coincideswithtransformationbutnotinflammation.A .No significantdifferenceinPKRexpressionisobservedbetweencasesof inflammation(n=16)andnormal(n=154)breasttissues. B .Both hyperplasia(n=31)anddysplasia(n=10)precanceroustissuespecimensdisplayincreasedPKRexpressioncomparedtonormaltissues (n=154).Statisticalsignificancewasdeterminedbyt-test.**Indicates p 0.01. doi:10.1371/journal.pone.0046040.g002 RoleofPKRinBreastCancer PLOSONE|www.plosone.org5September2012|Volume7|Issue9|e46040

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Figure3.IncreasedlevelsofphosphorylatedPKRarepresentinbreastcancercelllines.A .Westernblottingforphospho-T451-PKRin breastcancer(MCF7,T-47D,MDA-MB-231)and‘‘normal’’breastepithelial(MCF10A)celllinesdemonstratesincreasedbasallevelsof‘‘activated’ ’PKR presentinbreastcancercelllinescomparedtonormalbreastcells. B .WesternblotsshowingthatPKRlevelissignificantlydecreasedinbreastcancer celllinesthatstablyexpresssiRNAspecifictoPKR(siPKR)comparedtocontrolsiRNA(siControl). C.–E .Breastcancercelllineswithdecreasedlevelsof PKRproliferateatthesamerateascontrolcells.Experimentsweredoneintriplicate,mean 6 SDisshown. F.–H .Breastcancercelllineswith reducedPKRexpressionbysiRNAknockdowndisplayreducedlevelofcellinvasioncomparedtocontrolcells.Briefly20,000cellswereaddedtothe upperchamberofatranswellinsertinserumfreemediumwhilemediumcontaining10%FBSwasplacedinthelowerchamber.After24hours, invadingcellsinthelowerchamberwerestainedandOD560measured.Experimentswererepeatedfourtimesandmean 6 SDwasgraphed. Statisticalsignificancewasdeterminedbyt-test.*Indicatesp 0.05,**indicatesp 0.01. doi:10.1371/journal.pone.0046040.g003 RoleofPKRinBreastCancer PLOSONE|www.plosone.org6September2012|Volume7|Issue9|e46040

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tion,augmentsDOX-inducedcelldeathandsuggeststhatthe PKR-eIF2 a signalingpathwayisimportantforDOXcytotoxicity inbreastcancer.SinceDOXhasbeenthebackboneofcurrent standardcombinationchemotherapyregimensfortreatingbreast cancer,weproposethatincreasedPKRinprimarybreastcancer vs.normaltissuemayrepresentapositiveprognosticbiomarkerfor responsetochemotherapyandcontributetoDOX’sfavorable therapeuticindexwhenusedtotreatbreastcancer. Figure4.BreastcancercelllineswithreducedPKRarelesssensitivetodoxorubicinorH2O2. Breastcancercelllinesstablyexpressing eithersiRNAtoPKR(siPKR)orcontrolsiRNA(siControl)weretreatedwithincreasingconcentrationsofdoxorubicin(DOX)orH2O2for48hours. ViabilitywasmeasuredbyTrypanbluedyeexclusionassay.Experimentswererepeatedintriplicateandmean 6 SDgraphed. A .MCF7celllines treatedwithDOX. B .MCF7celllinestreatedwithH2O2. C .T-47DcelllinestreatedwithDOX. D .T-47DcelllinestreatedwithH2O2. E .MDA-MB-231cell linestreatedwithDOX. F .MDA-MB-231celllinestreatedwithH2O2. doi:10.1371/journal.pone.0046040.g004 RoleofPKRinBreastCancer PLOSONE|www.plosone.org7September2012|Volume7|Issue9|e46040

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Figure5.PKRactivityandexpressionpromotessensitivitytodoxorubicin.A .Breastcancercelllinesweretreatedwitheither2.5 m M doxorubicin(DOX)orco-treatedwith2.5 m MDOXandincreasingconcentrationsofPKRinhibitor(PKRI)for48hours.Cellviabilitywasmeasuredby RoleofPKRinBreastCancer PLOSONE|www.plosone.org8September2012|Volume7|Issue9|e46040

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WhileitisnotyetclearhowPKRexpressioncanbeelevatedin primarybreastcancertissuesorwhyincreasedPKRinsuch untreatedmalignanciesapparentlyfailstohindertheircellular growth,wespeculatethatPKRisinalatent,inactivatedstatein breastcancertissue.Thus,upregulationofPKRexpressioninand ofitselfmaynotbysufficienttobringaboutgrowthinhibition. Additionalselectivestresses(i.e.chemotherapysuchasdoxorubicin)mayneedtobeappliedtobreasttumorcellstoeffectPKR activationwithinhibitionofnewproteinsynthesisandenhanced apoptosis.Insupportofthis,breastcancercelllineswithreduced PKRexpressionbysiRNAknockdowndisplaynochangeinthe rateofcellproliferationundernormalgrowthconditions comparedtocontrolcells.Alternatively,PKR’santi-proliferative functionmaybesuppressedbysomeadditionaluncharacterized cellularmechanism(s)suchascompensatorychangesingene expression.Insupportofthis,ithasbeenreportedthatinaddition toincreasedPKRexpression,breastcancercelllinesmayexpress increasedP58andeIF2Bthatcouldenablethesecellsto circumventthegrowthinhibitioneffectofincreasedPKR expressionand/oreIF2 a phosphorylation.[38]Furtherstudies willbenecessarytofullyelucidatethefeaturesofbreastcancer cellsthatpromoteincreasedPKRexpressionwhilecircumventing PKR’sproapoptoticfunction. BreastcancercelllineswithreducedPKRdisplayedadelayin eIF2 a phosphorylationandreducedapoptosisfollowingtreatment withDOX.Importantly,treatmentwithIFNtoincreasePKR expressionorwithsalubrinaltopromoteeIF2aphosphorylation restoresDOXcytotoxicityincellswithreducedPKR.Furthermore,ourdataindicatethatinhibitionofPKRactivitywithasmall moleculecompoundreducesbreastcancercellsensitivitytoDOX. TakentogethertheseresultssuggestthatPKRactivityisnecessary toobtainthefullandpotenttherapeuticeffectofDOX.Thus, futuretherapeuticapproachesthatcanpromoteincreased expression/activationofPKRandphosphorylationofeIF2 a maybeaneffectivemodalityoftreatmentforbreastcancer patientswhosebreasttumorsdonotdemonstrateelevatedPKR.MaterialsandMethods CellLines,AntibodiesandOtherReagentsMCF10A(lot # 7690599),MCF7(lot # 7629688),T-47D(lot # 7516238),MDA-MD-231(lot # 57618051)cellswereobtained fromATCC(Manassas,VA).CellswerepropagatedinDulbecco modifiedEaglemedium(DMEM)supplementedwith10%fetal bovineserum(FBS),1%L-glutamineand1%Penicillin-Streptomycininahumidifiedincubatorat37 u Cand5%CO2(Life Technologies,Carlsbad,CA).Inaddition,mediumforMCF7cells contained5 m g/mlbovineinsulin(Sigma-Aldrich,St.Louis,MO). Doxorubicin(DOX),PKRinhibitorcompound(PKRI),paclitaxel andsalubrinalwerefromCalbiochem/EMDMillipore(Darmstadt,Germany).HydrogenperoxidewasfromSigma-Aldrich(St. Louis,MO).HumanIFN a wasfromR&Dsystems(Minneapolis, MN).Phospho-threonine451-specificPKRrabbitpolyclonal antibodywasfromInvitrogen/Biosource(GrandIsland,NY).Trypanbluedyeexclusionassay.Experimentswererepeatedintriplicateandmean 6 SDgraphed.Statisticalsignificancewasdeterminedbyt-test.* Indicatesp 0.05,**indicatesp 0.01. B .WesternblottingdemonstratesincreasedPKRexpressionfollowingtreatmentofbreastcancercelllines with500Units/mlInterferona (IFN a )for48hoursinbothcontrolsiRNA(siControl)andPKRsiRNA(siPKR)expressingcells. C.–E .IFN-inducedPKR expressionincreasesbreastcancercelllinesensitivitytodoxorubicin.BreastcancercelllinesstablyexpressingeithersiRNAtoPKR(siPKR)orc ontrol siRNA(siControl)weretreatedwithincreasingconcentrationsofdoxorubicin(DOX)inmediumcontaining500U/mlIFN a for48hours.Viabilitywas measuredbyTrypanbluedyeexclusionassay.Experimentswererepeatedintriplicateandmean 6 SDgraphed. F .PKRactivityisrequiredforIFNinducedsensitivitytoDOX.MCF7cellsweretreatedfor48hourseitherwith2.5 m MDOXandIFN a ,orco-treatedwithDOX,IFN a and1 m MPKRI.Cell deathwasmeasuredbyTrypanbluedyeexclusionassay.CellviabilitywasmeasuredbyTrypanbluedyeexclusionassay.Experimentswererepeated intriplicateandmean 6 SDgraphed.Statisticalsignificancewasdeterminedbyt-test.**Indicatesp 0.01comparedtoDOXtreatmentalone. doi:10.1371/journal.pone.0046040.g005 Figure6.PKRexpressionisrequiredforeIF2 a phosphorylation followingtreatmentofbreastcancercelllineswithdoxorubicin. BreastcancerlineswithreducedPKRexpression(siPKR)orcontrol cells(siControl)weretreatedwith2.5 m MDOXfortheindicatedtimes. Followingtreatment,cellswerecollectedandwesternblottingusing antibodyspecificforphosphoserine-51eIF2 a (P-eIF2 a ),eIF2 a ,cleaved PARP(cl-PARP),PKRoractinwasperformed.A.WesternblotofMCF7 cellsfollowingtreatmentwithDOXindicatescellswithreducedPKR haveareducedrateofeIF2 a phosphorylationandPARPcleavage.B. WesternblotofMDA-MB-231cellsfollowingtreatmentwithdoxorubicinindicatescellswithreducedPKRhaveareducedrateofeIF2 a phosphorylationandPARPcleavage.C.WesternblotofT-47Dcells followingtreatmentwithdoxorubicinindicatescellswithreducedPKR haveareducedrateofeIF2 a phosphorylation. doi:10.1371/journal.pone.0046040.g006 RoleofPKRinBreastCancer PLOSONE|www.plosone.org9September2012|Volume7|Issue9|e46040

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Figure7.PhosphorylationofeIF2 a increasesbreastcancercelllinesensitivitytodoxorubicin.A .Westernblottingusingantibody specificforphosphoserine-51eIF2 a (P-eIF2 a ),eIF2 a oractindemonstratesincreasedeIF2 a phosphorylationinbreastcancercelllinesfollowing48 hourstreatmentwith20 m Msalubrinal. B .– D .BreastcancercelllineseitherexpressingPKRsiRNA(siPKR)orcontrolsiRNA(siControl)weretreated witheither2.5 m MDOX( + DOX)orco-treatedwith2.5 m MDOXandincreasingconcentrationsofsalubrinalfor48hours.Viabilitywasmeasuredby Trypanbluedyeexclusionassay.Experimentswererepeatedintriplicateandmean 6 SDgraphed.Statisticalsignificancewasdeterminedbyt-test.* Indicatesp 0.05,**indicatesp 0.01comparedtoDOXtreatmentalone. doi:10.1371/journal.pone.0046040.g007 RoleofPKRinBreastCancer PLOSONE|www.plosone.org10September2012|Volume7|Issue9|e46040

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PKRM02monoclonalantibodyclone1D11wasfromAbnova (TaipeiCity,Taiwan).Phospho-serine51-specificeIF2 a ,eIF2 a andcleaved-PARPantibodieswerefromCellSignalingTechnology(Beverly,MA).AntibodytoactinwasfromSantaCruz BiotechnologyInc(SantaCruz,CA).TissueMicroarrayIHCStainingandAnalysisTissuemicroarrays(TMAs)wereobtainedfromBiomaxUS (Rockville,MD)andstainedbyimmunohistochemistry(IHC) usingmonoclonalantibodytoPKR.Thefollowingarrayswere stained:BR722,BR1002,BR1003,BR1006,BR2082,and BN08013.Antibodyoptimizationandstainingwasperformedby theUniversityofFlorida’smolecularpathologycorefacility. ImagesoftheIHCTMAsectionsweredigitizedusingaScanscope digitalslidescannerandvisualizedwithImagescope(Aperio, Vista,CA).Threepathologists,blindedtoallcharacteristicsof samples,independentlyquantifiedPKRimmunoreactivity.IHC analysisscoresweredeterminedbytakingtheproductofthe estimatedstainingintensity(0fornegative,1forweak,2for moderate,or3forstrong)andpercentageoftissuestained (0%=0, 25%=1,25%–75%=2, 75%=3),givingarangeof possiblescoresbetween0and9.Scoresforreplicatecoreswere averagedtodetermineacompositescoreforeachcase.T-testwith F-testwasperformedusingGraphPadPrismversion5(GraphPad Software,SanDiegoCaliforniaUSA,www.graphpad.com).KnockdownofPKRExpressionbysiRNATransduction-readylentivirusparticlescontainingshRNAs specificforhumanPKRwereusedtoknockdownPKRexpression inMCF7,T-47DandMDA-MB-231cellsaccordingtothe manufacturer’sprotocol(SantaCruzBiotechnology,Inc., # sc36263).AGFP-expressingcontrollentiviruswasusedtomeasure transductionefficiencyandoptimizeconditions.Aftertransduction,stablecelllineswereisolatedbyselectionwith2 m g/ml puromycin.Efficiencyofknockdownwasevaluatedbywestern blotting.CellProliferation,ViabilityandInvasionMeasurementsCellproliferationandviabilityduringnormalgrowthor followingtreatmenteitherwithDOX,H2O2,orpaclitaxelfor theindicatedconcentrationsandtimesweremeasuredbyTrypan bluedyeexclusionassay.Viableanddeadcellswerecountedwith theaidofanAutoT4Cellometer(NexcelomBioscience, Lawrence,MA).Inaddition,TUNELassaywasperformedusing anAPO-BRDUkit(BDBiosciences,SaneJose,CA).Cellinvasion wasmeasuredusingaCytoSelect24-WellCellinvasionassay (Basementmembrane,colorimetric)fromCellBiolabs,Inc.(San Diego,CA).Briefly,20,000cellswereplatedintheupperchamber inserum-freemediumwhilemediumcontaining10%FBSwas placedinthelowerchamber.After24hours,invasivecellswere stainedandOD560nmmeasuredaccordingtothemanufacturer’s protocol.StatisticalsignificancewascalculatedbyT-testusing GraphPadPrismversion5.SupportingInformationFigureS1DecreasedPKRexpressioninbreastcancer celllinesdecreasessensitivitytodoxorubicinbutnot paclitaxel.A .CellswithreducedPKRexpressionareless sensitivetoDOX.CellviabilitywasmeasuredinMCF7cells expressingeithersiRNAspecifictoPKR(siPKR)oracontrol siRNA(siControl)atvarioustimesfollowingtreatmentwith5 m M DOXbyTrypanbluedyeexclusionassay.Experimentswere repeatedintriplicateandmean 6 SDgraphed. B .TUNELassay byflowcytometrysuggeststhatMCF7andT-47Dcellsexpressing PKRsiRNA(siPKR)displayreducedapoptosiscomparedto control(siControl)cellsafter24hourstreatmentwith5 m MDOX. Experimentswererepeatedintriplicateandmean 6 SDgraphed. Statisticalsignificancewasdeterminedbyt-test.*Indicates p 0.05. C .ReducedPKRexpressiondoesnotaffectsensitivity topaclitaxel.ViabilityofMCF7cellsexpressingeitherPKR (siPKR)orcontrol(siControl)siRNAafter24hourstreatmentwith increasingconcentrationsofpaclitaxelwasmeasuredbyTrypan bluedyeexclusionassay.Experimentswererepeatedintriplicate andmean 6 SDgraphed. D .Viabilityafter24hoursco-treatment with2.5 m MDOXandincreasingconcentrationsofpaclitaxelwas measuredbyTrypanbluedyeexclusionassay.Experimentswere repeatedintriplicateandmean 6 SDgraphed. (TIF)AcknowledgmentsWewouldliketothankAnnFuandtheUniversityofFlorida’smolecular pathologycoreforexcellenttechnicalassistance.Inaddition,K.K.was aparticipantintheUniversityScholar’sProgram,andT.H.was aparticipantintheHHMI-UFScienceforLifeProgram.Publicationof thisarticlewasfundedinpartbytheUniversityofFloridaOpen-Access PublishingFund.AuthorContributionsConceivedanddesignedtheexperiments:RLBWSM.Performedthe experiments:RLBALCTHKRKXL.Analyzedthedata:RLBWSM. 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