tumor resistance to radiotherapy Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated Neoplasia

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Neoplasia

journalhomepage:www.elsevier.com/locate/neo

Original Research

Targeting Na-H exchanger 1 overcomes nuclear factor kappa B-mediated tumor resistance to radiotherapy

Arang Son

^a,1

, Seoyeong Kang

^b,1

, Suha Choi

^a

, Sung-Won Shin

^a

, Yeeun Kim

^a

, Wankyu Kim

^b,∗

, Changhoon Choi

^a,∗

aDepartment of Radiation Oncology, Samsung Medical Center, Seoul 06351, South Korea

bDepartment of Life Sciences, Ewha Womans University, Seoul 03760, South Korea

a r t i c le i n f o

Keywords:

Radiation therapy Acquired radioresistance Na-H exchanger 1 NF- 𝜅B

a b s t r a ct

Intrinsicoracquiredradioresistanceoftenlimitstheefficacyofradiationtherapy(RT),therebyleadingtolocal controlfailure.CancerouscellshaveabnormalpHdynamicsduetohighmetabolicdemands,butitisunclearhow pHdynamicscontributetoradioresistance.Inthisstudy,weinvestigatedtheroleofNa-Hexchange1(NHE1), themajorintracellularpH(pH_i)regulator,inRTresponse.WeobservedthatRTincreasedNHE1expressionand modulatedpH_iinMDA-MB-231humanbreastcancercells.WhencombinedwithRT,pharmacologicalNHE1 inhibitionby5-(N-Ethyl-N-isopropyl)amiloride(EIPA)reducedpH_i andclonogenicsurvival.EIPAattenuated radiation-damagedDNArepair,increasingG2/Mcellcyclearrest.ThecombinationofEIPAandRTincreased apoptoticcelldeathwhiledecreasingphosphorylationofNF-𝜅Bp65.Similarly,theknockdownofNHE1in- creasedradiosensitivitywithlowerpH_i andincreasedapoptosis.Consistentwithinvitrodata,theEIPAplus RTinhibitedthegrowthofMDA-MB-231xenografttumorsinmicetoagreaterextentthaneitherEIPAorRT alone.EIPAabrogatedtheRT-inducedincreaseinNHE1andphospho-NF-𝜅Bp65expressionintumortissues.

SuchcoincidenceofincreasedNHE1level,pH_i,andNF-𝜅BactivationwasalsofoundinradioresistantMDA-MB- 231cells,whichwerereversedbyEIPAtreatment.BioinformaticsanalysisofRNAsequencingdatarevealedthat inhibitingNHE1reversedthreecoregenenetworksthatwereup-regulatedinradioresistantcellsandcorrelated withhighNHE1expressioninpatientsamples:NF-𝜅B,senescence,andextracellularmatrix.Takentogether,our findingssuggestthatNHE1contributestoRTresistanceviaNF-𝜅B-mediatedsignalingnetworks,andNHE1may beapromisingtargetforimprovingRToutcomes.

Introduction

EmergingdatasuggestthataberrantpHdynamicsisoneofthechar- acteristicsthatcancercells acquireadaptively [1,2]. Cancerouscells haveareversedpHgradient, withahigherintracellularpH(pH_i) of

≥7.2andalowerextracellularpH(pH_e)of6.7– 7.1,comparedtonor- malcells,whichhavelowerpH_iandhigherpH_e.Thisisprimarilydue toincreasedproliferativeandmetabolicactivitiesofcancercellswhen comparedtonormalcells.Indeed,variousnetacidextruders,including H⁺-ATPases,theNa⁺-H⁺exchangerNHE1,andthemonocarboxylate- H⁺effluxcotransportersMCT1andMCT4areupregulatedoractivated incanceroustissuestomaintainpHhomeostasis,andvarioustherapeu- ticstrategiestoexploitthispHdysregulationhavebeentested[3].

CancercellsbenefitfromthereversedpHgradientinproliferation, survival, metabolism,migration, and metastasis.Increased pH_i pro- motesproliferationasapermissivesignalforgrowthfactor-stimulated

∗Correspondingauthors.

E-mailaddress:chchoi93@gmail.com(C.Choi).

1 Theseauthorscontributedequally.

cell cycle progression, particularly the G2/M transition [4]. It also allows cancercellstoavoid apoptosis[5,6]. Aerobicglycolysis, also knownastheWarburgeffect,isanessentialmetabolicfeatureofcan- cer cells,andpHsensorslike phosphofructokinase-1 areinvolvedin pH_i-regulatedmetabolism[7].AnotherexampleofpH-regulatedcan- cer cell behavioris directed cellmigration. Migrating cells remodel actinfilamentsandcell-substrateadhesion,whichisregulatedbyin- tracellular pHsensors such as focal adhesion kinase [8]. Decreased pH_e not only causes melanoma cells to migrate faster by modulat- ingintegrin-extracellularmatrixattachments[9]butitalsoisrequired forinvadopodiummaturationandmatrixmetalloproteinaseactivation, whichfacilitatesinvasionandmetastasis[10].

AmongthemanypHregulators,NHE1(SLC9A1),amemberofthe solutecarrierfamily9,playsacriticalroleinpH_imaintenance.NHE1 expressionoractivityiselevatedinvarioustypesofcancers,including gastriccancer[11],ovariancancer[12],andgliomatissues[13]andis

https://doi.org/10.1016/j.neo.2022.100862

Received10October2022;Receivedinrevisedform30November2022;Accepted30November2022

1476-5586/© 2022TheAuthors.PublishedbyElsevierInc.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

A. Son, S. Kang, S. Choi et al. Neoplasia 35 (2023) 100862

associatedwithpoorprognosis.PreviousresearchhasshownthatNHE1- deficienthumanbladdercarcinomacellsandtriple-negativebreastcan- cer cells grow poorly in immune-compromised mice, implying that NHE1isrequiredfortumorgrowth[14,15].Pharmacologicalinhibition ofNHE1demonstratesanti-cancerefficacyinpancreaticcancer[16], andfibrosarcoma[17]mousexenograftmodels.Furthermore,NHE1in- hibitionre-sensitizesdrug-resistantcancer[15,18]andworkssynergis- ticallywithtargetedtherapieslikeerlotinib[19]andimmunotherapy [20].

Radiationtherapy(RT)isoneofthemosteffectivetreatmentmodal- itiesforsolidcancer,butitislimitedbyintrinsicoracquiredradioresis- tance.Severalpreclinicalstudieshavebeenconductedtoinvestigatethe rolesofnetacidexcludersinRTresponse.Interruptinglactatetransport byinhibitingeitherMCT1ormitochondrialpyruvatecarrierincreased radiosensitivityinsmallcelllungcancerandcervicalcancerxenografts [21,22].CarbonicanhydraseIX(CA9),whichcontributestoacidicpH_e, is linkedtotumor hypoxia andradioresistance. Itsinhibition makes breastcancer,renalcellcarcinoma,andcoloncarcinomasensitiveto RT[23,24].NHE1inhibitiondiminishesthepH_i-regulatingcapacityof fibroblastandcancercells,increasingradiosensitivity[24],buttheun- derlyingmechanismsremainunknown.Thegoalofthisstudywasto investigatethemechanismofNHE1-regulatedradioresistance.

Materialsandmethods Cellculture

MDA-MB-231humantriple-negativebreastcancer(TNBC)cellline wasobtainedfromtheAmericanTypeCultureCollection(ATCC,Manas- sas,VA,USA),andAsPC-1humanpancreaticcancercelllineswereob- tainedfromKoreanCellLineBank(SeoulNationalUniversity,Seoul,Ko- rea).MDA-MB-231andAsPC-1cellswereculturedinDulbecco’sModi- fiedEagleMedium(DMEM)supplementedwith10%fetalbovineserum (FBS),1×antibiotic-antimycoticsolution(AA,ThermoFisherScientific, Waltham,MA,USA)and25mMHEPES(Gibco,Carlsbad,CA,USA).The radioresistantMDA-MB-231cellline(MDA-MB-231RR)wasestablished byexposingtheparentalMDA-MB-231celllinetocumulativedoses(to- talof40Gy)ofradiation.Initially,adailydoseof2Gywasdeliveredto thecellsover5daysfor2weeks.Afterrestingfor2weeks,thecellswere collectedandplatedatlowdensity.Thecellswereirradiatedwithan additionaldailydoseof5Gytwiceaweekfor2weeksandthesurviving cellswerepooled.Acquiredradioresistancewasdeterminedbyclono- genicsurvivalassay.Cultureswerekeptinahumidifiedatmosphereof 95%air/5%CO₂at37°C.

Reagentsandantibodies

5-(N-Ethyl-N-isopropyl)amiloride(EIPA)waspurchasedfromTocris Bioscience (Bristol,UK).Anti-phospho-H2AX (Ser139), anti-phospho- ATR(Ser428),anti-phospho-Chk2(Thr68),anti-NF-𝜅Bp65,andanti- cleavedPARPantibodieswerepurchasedfromCellSignalingTechnol- ogy(Danvers,MA,USA).Anti-𝛽-actinantibodieswerepurchasedfrom Sigma Aldrich (St. Louis, MO, USA).Anti-NHE1 andanti-MCT1 an- tibodieswerepurchasedfromSantaCruz Biotechnology(SantaCruz, CA,USA).Anti-phospho-NF-𝜅Bp65(Ser529)antibodieswerepurchased fromBDbioscience(Franklin,NJ,USA).

Cellviabilityassay

CellviabilitywasdeterminedusingaCCK-8(CellCountingKit-8,Do- jindoLaboratories,Kumamoto,Japan)assay.MDA-MB-231andAsPC-1 cellswereseededintoa96-wellplateat1×10⁴cells/wellandtreated withvariousconcentrationsof EIPA.Cell viabilitywas calculatedas a percentagerelativeto theuntreated controlbymeasuring theab- sorbanceat450nmwithaSpectraMaxi3microplatereader(Molecular Devices,Sunnyvale,CA,USA).

Irradiation

Asingle-cellsuspensionofexponentiallygrowingcellswasseeded oncelldishesadaybeforeirradiation.Atasourcesurfacedistanceof 100cmandafieldsizeof30×30cm,thedisheswereplacedbeneatha 2cmthicksolidwaterphantom.X-raybeamsweredeliveredatadose rateof3.96GyperminusingaVarianClinac6EXlinearaccelerator (VarianMedicalSystems,PaloAlto,CA,USA).TheabsoluteX-raydose wasverifiedtoa1%accuracyinaccordancewithTG-51.

Clonogenicassay

The clonogenic survival assaywas used to determineradiosensi- tivity. Cellswereincubatedfor7-14daysafterirradiation.Surviving colonieswithmorethan50cellswerecountedafterbeingstainedwith 0.5 % crystal violet.The survival fraction was calculatedas aratio of theplatingefficiency(number ofsurviving coloniestonumberof platedcells)oftheirradiatedcellstomock-irradiatedcells.Thedose- response curvesweredrawnusing GraphPad Prism9.3.1(GraphPad Software,LaJolla,CA,USA)andwerefittedbyalinear-quadraticequa- tion[SF=exp(−𝛼D−𝛽D²);SF,survivalfraction;D,absorbeddose].

Thedoseenhancementfactor(DEF)ofEIPAistheratiooftheradiation dosesresultingin10%survivalcomparingcontrolsamples.

Bioluminescenceassay

Caspase-Glo3/7reagentassay(Promega,Madison,WI, USA)was performedaccordingtothemanufacturer’sinstructions.Briefly,MDA- MB-231cellswereseededat1×10⁴cells/wellintoaclearbottomwhite 96-wellplate,thenincubatedwith20μMEIPA,followedbyIR.After48 h,100μLofCaspase-Glo3/7reagentwasadded,andluminescencewas measuredwithaSpectraMaxi3microplatereader(MolecularDevices, Sunnyvale,CA,USA).

For measuring NF-𝜅B activity, cells were transfected with an NF-𝜅Bpromoter-luciferaseplasmid(pGL4.32[luc2P/NF-𝜅B-RE/Hygro], Promega)andRenillaluciferaseplasmid(pRL-TK,Promega)usingLipo- fectamine3000(ThermoFisherScientific)accordingtothemanufac- turer’sinstructions.EIPAwasappliedtotransfectedcellsfor24h.Lu- ciferaseactivitywasmeasuredusingaDual-Gloluciferaseassaysystem (Promega).

IntracellularpHmeasurement

IntracellularpHwasdeterminedbyusingthepHrodoGreenAMin- tracellularpHindicator(ThermoFisherScientific)accordingtotheman- ufacturer’sinstructions.Briefly,cellswereseededat2×10⁴cells/well intoaclearbottomblack96-wellplate.CellswerewashedwithLiveCell ImagingSolution(LCIS)andstainedfor30minwiththepHrodoAMEs- terstainingsolution.Thefluorescenceintensitywasdeterminedusinga SpectraMaxi3microplatereaderwithexcitation/emissionwavelengths of509/533nm.ApH_ititrationcurvewasobtainedbyincubatingwith pH_icalibrationbufferscontainingnigericinandvalinomycin(pH5.5, 6.5,and7.5).

SmallinterferingRNA(siRNA)transfection

Cells (1×10⁶) weretransfected with10 nMsiRNAdesignedfor NHE1knockdown(sc-42650,SantaCruzBiotechnology)byusingLipo- fectamine RNAi MAX reagent (Thermo Fisher Scientific, Waltham, MA,USA),accordingtothemanufacturer’sinstructions.Non-targeting siRNA(sc-37007)wasusedforcontrolexperiments.

Westernblotanalysis

CellswerelysedinmodifiedRIPAbuffer(20mMTris(pH8.0),137 mMNaCl,10%glycerol,1%NonidetP-40,10 mMEDTA,100mM

NaF,1mMphenylmethylsulfonylfluorideand10mg/mLleupeptin),and thepelletwasremovedafter15minofcentrifugationat13,000rpm.

ProteinconcentrationwasdeterminedusingtheBio-Radproteinassay reagent(Bio-Rad,Richmond,CA,USA)accordingtothemanufacturer’s instructions.Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis (SDS-PAGE)wascarriedoutonequalamountsofprotein.Proteinswere separatedandtransferredtonitrocellulosemembranes(Bio-Rad);blots wereblockedovernightwith5%skimmedmilkinPBSat4°Cbefore beingtreatedwithprimary antibodiesovernight. Proteinbandswere detectedusingtheAmershamenhancedchemiluminescencedetection reagents(GEHealthcare,Piscataway,NJ,USA).

Flowcytometry

Forapoptosisdetection,harvestedcellswerestainedwithannexin V-FITC(BDPharmingen,SanDiego,CA,USA),and2μg/mLpropidium iodide(PI)wasaddedtotheannexinVbindingbuffer(10mMHEPES, pH7.4,140mMNaCl,2.5mMCaCl₂)inthedarkfor15minat37°C.

Thenumberofapoptoticcells wasquantifiedusing aBDFACSVerse flowcytometerandtheBDFACSuitesoftware.Forthecellcycleanal- ysis,harvestedcellswerefixedwithpre-chilled70%ethanolandwere incubatedwith1mg/mLRNaseand50μg/mLPIinthedarkfor30min at37°C.

Immunofluorescence

Cellsculturedoncoverglasseswerefixedwith4%formaldehydeand permeabilizedwith0.01%TritonX-100.After30minofblockingwith 2%FBS,cellswereincubatedwithprimary𝛾-H2AXantibodyfor1h, followedbyDAPI(Sigma-Aldrich)andAlexaFluor-488-conjugatedsec- ondaryantibody(LifeTechnologies)for 30min.Afterthat, thecells werewashed, mountedwitha fluorescentmounting medium (Dako, Carpinteria,CA,USA),andexaminedunderafluorescencemicroscope (ZeissObserverD1;CarlZeissCo.,Ltd.Carpinteria,CA,USA)

Animalmodels

Allanimalprocedures wereconductedin accordancewithappro- priate regulatorystandardsunder a protocol(ID: 20200909002; ap- provaldate: 2020-09-01) approvedbythe InstitutionalAnimal Care andUseCommittee(IACUC)oftheSamsungBiomedicalResearchIn- stitute(SBRI).TheSBRIisanaccreditedfacilityoftheAssociationfor AssessmentandAccreditationofLaboratoryAnimalCareInternational (AAALACInternational)andabidesbytheInstituteofLaboratoryAni- malResources(ILAR)guidelines.

FortheMDA-MB-231tumor model,6–7-week-old femaleBALB/c micewerepurchasedfromOrientBio(Gapyeong,Korea).Cells(5×10⁶ cells/50μLPBS)wereinjectedsubcutaneouslyintotherighthindleg.

Tumorvolumesweremeasuredevery3dayswithcalipersandcalcu- latedaccordingtothefollowingformula:volume =width² ×length

÷ 2.Whenthemeantumorvolumereached80–120mm³,micewere randomlyassignedtooneoffourgroups.EIPA(12mg/kg)wasgiven intraperitoneallythreetimesaweekforthefirsttwoweeks,thentwice aweekforthenexttwoweeks.Thetumorwasirradiatedwith6GyX- raytotherighthindlegtwohoursafterdrugtreatment.Themicewere intraperitoneallyanesthetizedwith30mg/kgofZoletil(Virbac,Carros, France)and10mg/kgofRompun(Bayer,Leverkusen,Germany).

TUNELandimmunohistochemistry

TheterminaldeoxynucleotidyltransferasedUTPnickendlabeling (TUNEL)assaywasusedtodetectapoptosisintumortissues.Slicesof paraffin-embeddedtissuesweredeparaffinizedinwaterandplacedin 3%H₂O₂atroomtemperaturefor10min.TheApopTag® PeroxidaseIn SituApoptosisDetectionKit(Millipore)wasusedaccordingtothemanu- facturer’sinstructions.TUNEL-positivecellswereimagedandquantified

usingtheimageScopesoftwareonanAperioScanScopeATinstrument (LeicaBiosystems,BuffaloGrove,IL,USA).

Forimmunohistochemistry, theparaffin-embedded tissuesections weredeparaffinizedthreetimesinxylenefor15minbeforebeingre- hydrated.ImmunostainingforNHE1andphospho-NF-𝜅Bp65wasper- formed using the Bond‐MaxTM polymer refine detection kit (Vision Biosystems,Melbourne,Australia).Antigenretrievalwasperformedin ER1bufferat97°Cfor20min.For10min,3%H₂O₂wasusedtoinhibit endogenousperoxidaseactivity.Theprimaryantibodyincubationwas performedfor15minatroomtemperature.

Statistics

StatisticalanalysiswasperformedusingGraphPadPrism9.3.1.The statisticalsignificanceofdifferencesamonggroupswascalculatedwith one-wayortwo-wayANOVAwithTukey’smultiplecomparisonspost- hoctest.Allp-values<0.05wereconsideredstatisticallysignificant.

RNA-sequencingandpreprocessing

MDA-MB-231-RRcellsweretreatedwith20or50μMEIPAandhar- vestedafter24h.TotalRNAwasextractedusingTRIzolreagent(Thermo Fisher Scientific).Transcriptomedatawasproducedby IlluminaNo- vaSeq 6000andTruSeqStranded mRNAlibraryKit.Thereads were alignedtotheGRCh38referencegenomeusingSTAR(v2.7.1a)[25],and geneexpressionwasquantifiedwithRSEM(v1.3.1)[26].Onlyprotein- codinggeneswereusedforsubsequentanalysis.

Differentialexpressionanalysis

DifferentialexpressionanalysiswasperformedusingDESeq2[27]. Forwild-typeandradioresistancesamples(WTversusRRgroup),anal- ysiswasperformedbybatchandtheresultingdifferentiallyexpressed genes(DEGs)wereaggregated(foldchange>2,FDR<0.01).ForEIPA- treatedsamples(EIPAversusRRgroup),differentthresholdswereap- pliedforeachdrugconcentration(foldchange>1.5andFDR<0.3for 20μM,foldchange>2,andFDR<0.01for50μM).Pathwayandtran- scriptionfactoranalysiswereperformedusingahypergeometrictest.

GenesetswerecollectedfromMSigDB(v7.4)[28]andHarmonizome [29].ThegenenetworkwasconstructedfromSTRINGprotein-protein interactiondatabase[30]andvisualizedusingCytoscape[31].Theden- sityofgenenetworkwasdefinedbythefollowingformula:

𝑁𝑒𝑡𝑤𝑜𝑟𝑘𝑑𝑒𝑛𝑠𝑖𝑡𝑦= 𝐸

𝑁(𝑁−1) 2

WhereErepresentsthenumberoftotaledgesinthenetworkandN representsthenumberofnodes.

Thecancergenomeatlas(TCGA)dataanalysis

The expression data of BRCA patients in TCGA (http://

cancergenome.nih.gov/) was downloaded from the NCI Genomic DataCommons(GDC)DataPortal[32]andthepartialannotationdata wasfromtheGDCLegacyArchive.

TheTNBCpatientswereselectedbyexpression(FPKM)ofERBB2, ESR1,andPGR.TheFPKMcut-off oftriplegeneswasdecided(ERBB2

<30,ESR1<10,PGR<10)basedon127TNBCpatientswhosetriple geneswereannotatedasNegativebyImmunohistochemistry(IHC).Us- ingthesecriteria,239outof1222BRCApatientswereconservatively assumedasTNBC.Thesepatientseitherreceivedradiotherapy(RT,n= 123)ordidnot(noRT,n=81).

GeneSetenrichmentanalysis(GSEA)[33]foreachindividualsam- plewasperformedusingfgseapackage[34]withgenesetenrichedin MDA-MB-231RRmodels.Samplesweresortedbythesumofnormalized enrichment scores(NES)andclassified asNES-high and–low groups (100eachforTNBC,60forRT,and40fornoRT).ComparisonofNHE1

A. Son, S. Kang, S. Choi et al. Neoplasia 35 (2023) 100862

Fig.1.IonizingradiationincreasestheexpressionoftheNa-Hexchanger1(NHE1)andaltersintracellularpH(pH_i).(A)Westernblotanalysisshowedanincreasein NHE1expressionfollowingX-rayirradiationwith6Gy.InMDA-MB-231cells,sampleswerecollectedattheindicatedtimes(h)afterirradiation.𝛽-actinwasusedasa loadingcontrol.(B)NHE1expressionquantification.(C)ApH_ititrationcurvewiththefluorometricpHrodoGreenprobe.(D)EIPA,5-(N-Ethyl-N-isopropyl)amiloride, aselectiveNHE1inhibitor,decreasedpH_iconcentration-dependently.(E)ModulationofpH_ibyionizingradiation.(F)CombiningEIPAandX-raysreducedpH_ieven furtherinMDA-MB-231cells.Thedatarepresentthemean±SD(n=3).^∗p<0.05,^∗∗p<0.01,^∗∗∗p<0.001.

expressionbetweengroupswasperformedusingtheWilcoxontest,and survivalanalysiswasperformedusingthesurvivalpackage,whichesti- matesthroughthelog-ranktest.

Results

PharmacologicalinhibitionofNHE1decreasessteady-stateintracellularpH inMDA-MB-231cells

TodeterminetheroleofpH_iinradiationresponse,wefirstinves- tigatedhowionizingradiationaffectsNHE1,akeyregulatorofcellu- larpHhomeostasis.WesternblotanalysisshowedthattheNHE1level increased3.6-fold24hpost-irradiation(Fig.1AandB).siRNAtreat- mentconfirmedthespecificityofNHE1immunoblotting.Wethenused apH-sensitivefluorescencedye,pHrodoGreen,tomeasureradiation- mediatedchanges in steady-statepH_i.A pHtitrationcurvewas cre- atedusingthreedifferentpHcalibrationbuffers,demonstratinglinear- itywithinapHrangeof5.5to7.5(Fig.1C).TreatmentwithanNHE1 inhibitor 5-(N-Ethyl-N-isopropyl)amiloride(EIPA)decreased pH_i in a concentration-dependentmanner(Fig.1D).Low-doseradiation,suchas 2Gy,increasedpH_ifrom7.4to7.7(Fig.1E).However,high-dosera- diationabove6GydecreasedpH_i(Fig.1E),anditscombinationwith EIPAdecreasedpH_ievenmore(Fig.1F).

PharmacologicalinhibitionofNHE1increasesradiationsensitivityin MDA-MB-231andAsPC-1cells

EIPAtreatmentmodestlydecreasedMDA-MB-231andAsPC-1cell viabilityinadose-dependentmannerat48h(Fig.2A).Weperformed

aclonogenicsurvivalassaytoseehowinhibitionofNHE1affectedra- diosensitivity.Treatmentwith5μMEIPAsignificantlydecreasedthe platingefficiencyofMDA-MB-231cells(from52%to35%,p<0.001).

Since onlyafew coloniesgrowat concentrationshigherthan5μM, we chose5μMfortheclonogenicsurvivalassay. Pre-treatmentwith EIPA significantly reduced clonogenic survival of MDA-MB-231 and AsPC-1 cells in response toirradiation (DEF₁₀ = 1.43and 1.20for MDA-MB-231andAs-PC-1cells,respectively;Fig.2B),indicatingthat EIPA exertsa radiosensitizingeffect. EIPAwas alsofoundtohavea cytotoxic effect on breast andpancreaticcancer spheroids grownin 3Dculture (Fig.S1AandB).EIPAinhibitedthegrowthofMDA-MB- 231andAsPC-1spheroidsevenmorewhencombinedwithradiation (Fig.S1C).

TobetterunderstandhowEIPAexertsaradiosensitizingeffect,we investigateditsimpactonradiation-inducedDNAdamageresponseand cellcycleinMDA-MB-231 cells.Weused20μMEIPAbecauseitsig- nificantlyreducedpH_i(Fig.1D).Time-dependentanalysisrevealedthat theexpressionof𝛾-H2AX,aDNAdouble-strandbreak(DSB)marker, increased30minafterirradiationanddecreased24hlater(Fig.2C).

Radiation-induced expressionof𝛾-H2AX andphospho-CHK2was de- layedbypre-treatmentwith20μMEIPA(Fig.2C).Immunofluorescence revealedthatthenucleiofcellsco-treatedwithEIPAandradiationfor 24hhadmorefociof𝛾-H2AXthanradiationalone(Fig.2D).Cellcycle analysisshowedthatradiationcausedG2/Mphasearrest,whichwasex- acerbatedbytheadditionof20μMEIPA(Fig.2EandF).Thesefindings suggestthatinhibitingNHE1maymakecancercellsmorevulnerable toradiationbydelayingtherecognitionofDNAdamageandcellcycle re-entry.

Fig.2. NHE1inhibitionincreasesradiationsensitivityinMDA-MB-231andAsPC-1cells.(A)EffectofEIPAonMDA-MB-231andAsPC-1cellviability.TheCCK-8 assaywasperformedtoassesscellviability.(B)ClonogenicsurvivalassayofMDA-MB-231andAsPC-1cellstreatedwithorwithout5μMEIPAandthenexposedto 2,4,and6GyX-rays.Thedatarepresentthemean±SD(n=3).^∗∗p<0.01,^∗∗∗p<0.001.(C)WesternblotanalysisshowedthatEIPAattenuatedDNAdamage response.𝛽-actinwasusedasaloadingcontrol.(D)Representativeimmunofluorescenceimagesof𝛾-H2AXfociinMDA-MB-231nuclei.Cellsweretreatedwith20μM EIPAfor3hbeforebeingexposedto4GyofX-rays.After24h,thecellswerefixedandstained.(E)EIPAaugmentedradiation-inducedG2/Marrest.Representative histogramswerepresented.MDA-MB-231cellswereincubatedwith20μMEIPAfor3hbeforebeingexposedto4Gyirradiation.Cellcycleprogressionwasexamined 24hafterirradiation.(F)Cellcyclephasequantification.Thedatarepresentthemean±SD(n=3).

PharmacologicalinhibitionofNHE1enhancesradiation-inducedapoptosis inMDA-MB-231cells

WenextdeterminedtheeffectoftheNHE1inhibitoronradiation- inducedapoptosis,oneoftheradiation-inducedcelldeathmodes.Flow cytometrywithAnnexinV/PIco-stainingrevealedthathighconcentra- tionsofEIPA(above50μM)increasedapoptoticcelldeath(Fig.3Aand B).WhencomparedtoX-rayalone,thecombinationof20μMEIPAand4 GyofX-raysignificantlyincreasedapoptosis(p<0.01)comparedwith X-rayalone(Fig.3C).WithEIPAorX-ray,thecaspaseGlo3/7assay showedadose-dependentincreaseincaspase3/7activity(Fig.3Dand E).Whencombinedwith4Gyor6GyofX-ray,pre-treatmentwith20 μMEIPAincreasedcaspase3/7activityevenmorethanshamtreatment (Fig.3F).Westernblotanalysisconfirmedanincreaseinapoptosisinthe cellsco-treatedwithEIPAandX-ray,asdeterminedbycleavedPARP expression(Fig.3G).Thecombinedtreatmentreducedtheexpression oftheanti-apoptoticproteinMCL-1whileincreasingtheexpressionof pro-apoptoticBAK(Fig.3G).Furthermore,radiationincreasedthephos-

phorylationofNF-𝜅Bp65,whichisassociatedwithpro-survivalactiv- ity,whichwasreversedbyEIPAtreatment(Fig.3GandH).Thesedata suggestthatinhibitingNHE1promotesradiation-inducedapoptosisby suppressingpro-survivalsignaling.

KnockdownofNHE1increasesradiationsensitivityinMDA-MB-231cells

Itwasrecentlyreportedthatlong-termtreatmentwithEIPAhasan anticancereffectin3Dculture,independentofNHE1[35].Toverify whethertheradiosensitizingofEIPAwasNHE1-dependent,wesilenced NHE1expressioninMDA-MB-231cellsusingRNAinterference.Aswith EIPAtreatment,NHE1depletionwithsiRNAdecreasedpH_i(Fig.4A).

NHE1knockdownsignificantlyreducedclonogeniccellsurvivalinre- sponsetoradiation(Fig.4B).Immunoblottingshowedthattheexpres- sionof 𝛾-H2AXwasdelayed inNHE1-knockdown cells,compared to cellstransfectedwithcontrolsiRNA(Fig.4C).Radiation-inducedapop- tosiswasincreasedsignificantlymoreinNHE1-knockdowncellsthanin controlsiRNAcells(p<0.001),asdeterminedbyflowcytometryanaly-

A. Son, S. Kang, S. Choi et al. Neoplasia 35 (2023) 100862

Fig.3. NHE1inhibitionenhancesradiation-inducedapoptosisinMDA-MB-231cells.(A)Flowcytometrydiagramsofapoptoticcellsco-stainedwithPIandFITC- annexin.(B)Quantificationresultsshowedthat50μMEIPAinducedapoptoticcelldeath.(C)EIPAincreasedradiation-inducedapoptosis.(D)TheCaspase-Glo3/7 assaydemonstratedthat50μMEIPAinducedcaspase-dependentapoptosis.(E)Radiationincreasedthecaspase3/7activity.(F)EIPAincreasedradiation-induced caspase3/7activity.(G)WesternblotanalysisshowedthatEIPAincreasedradiation-inducedapoptoticcelldeath,asmeasuredbyincreasedPARPcleavage.𝛽-actin wasusedasaloadingcontrol.(H)QuantificationofphosphorylatedNF-𝜅Bp65usingImageJ.Thebandintensitywasnormalizedto𝛽-actin.Alldatarepresentmean

±SD(n≥3).^∗p<0.05,^∗∗p<0.01,^∗∗∗p<0.001.

siswithAnnexinV/PIco-staining(Fig.4DandE).TreatmentwithNHE1 siRNAabolishedtheradiation-inducedNHE1expressionandincreased theexpressionof𝛾-H2AXandcleavedPARP(Fig.4F).Thesedatasug- gestthatEIPAradiosensitizescancercellsdependentlyonNHE1.

InhibitionofNHE1augmentsradiation-inducedtumorgrowthdelayinan MDA-MB-231xenograftmousemodel

Totestwhetherinhibition ofNHE1 exerts its radiosensitizingef- fectinvivo,weinoculatedMDA-MB-231cellsintherighthindlegsof BALB/cnudemiceandtreatedthexenografttumorswithradiationand EIPA(Fig.5A).EitherEIPAor6GyofX-rayirradiationattenuatedthe growthofMDA-MB-231tumors,althoughthedifferencewasnotstatis- ticallysignificant(Fig.5B).ThecombinationofradiationandEIPAsig- nificantlyinhibitedtumorgrowthcomparedtoshamtreatmentorradi- ationalone(Fig.5B).Bodyweightdidnotchangeduringthetreatments, andnovisibletoxiceffectwasseen(Fig.5C).Tumorswereharvested 54daysafterirradiationforfurtheranalysis.TheTUNELassayoftu- mortissuesrevealedthateitherEIPAorIRaloneslightlyincreasedthe

apoptoticcellpopulation,whichwasmarkedlyincreasedbythecom- binationtreatment(Fig.5DandE).Next,wetestedhowradiationor EIPAaffectsNHE1andp-NF-𝜅Bp65levelsinthetumortissuesusing immunohistochemical(IHC)staining.Asingledoseofradiationsignifi- cantlyincreasedtheabundanceofNHE1andphospho-NF-𝜅Bp65inthe tumortissues,whichwassuppressedbyEIPAco-treatment(Fig.5F,G, andH).OurdataindicatethatNHE1inhibitionfurtherattenuatedthe growthoftheirradiatedtumors,whichmayberelatedtoadecreased pro-survivalsignaling, includingNF-𝜅Bwith increasedapoptoticcell death.

RadioresistantMDA-MB-231cellshaveincreasedNHE1and phosphorylationofNF-𝜅B

To determine whether NHE1 contributes to acquired radioresis- tance,wecreatedaradioresistantMDA-MB-231celllineandinvesti- gatedNHE1-mediatedsignaling.Apoolofsurvivedcellswascollected andnamed MDA-MB-231RRaftera totalof 40 Gywas delivered to thecells(fivefractionsof2Gyandfourfractionsof5Gy)(Fig.6A).

Fig.4. KnockdownofNHE1increasedradiationsensitivityinMDA-MB-231cells.(A)NHE1knockdowndecreasedpH_i.(B)NHE1knockdownreducedclonogenic survivalinresponsetoX-rayirradiation.(C)Westernblotanalysisshoweddelayedexpressionof𝛾-H2AXinNHE1-knockdowncells.𝛽-actinwasusedasaloading control.(D)RepresentativeflowcytometrydiagramshowinginductionofapoptosisinirradiatedNHE1-knockdowncells.(E)Quantificationdatashowingenhance- mentofradiation-inducedapoptosisbyNHE1knockdown.Alldatarepresentmean±SD(n≥3).^∗∗p<0.01,^∗∗∗p<0.001.(F)Westernblotanalysisshowedthat NHE1siRNAdepletedNHE1expression,increasedradiation-inducedexpressionofcleavedPARPand𝛾-H2AX,anddecreasedMCL-1expression.Thesampleswere prepared72hpost-irradiation.

TheclonogenicassayconfirmedthatMDA-MB-231RRcellsweresignifi- cantlymoreradioresistantthanparentalcells(Fig.6B).MDA-MB-231RR cellshadincreasedpH_icomparedtotheMDA-MB-231cells(Fig.6C).As withincreasedpH_i,radioresistantcellsexpressedhigherlevelsofNHE1 andphospho-NF-𝜅Bp65butnotMCT1(Fig.6D).MDA-MB-231RRcells weremoresensitivetoEIPAthantheirparentalcellsinacolonyforma- tionassay(Fig.6E).EIPAtreatmentreducedclonogenicsurvivalofir- radiatedMDA-MB-231RRcells,indicatingthatEIPAre-sensitizedMDA- MB-231RRcellstoradiation(Fig.6F).EIPAtreatmentreducedNHE1 andphospho-NF-𝜅Bp65levelsinMDA-MB-231RRcells(Fig.6G).EIPA treatmentalsoinhibitedtheactivityofNF-𝜅B-dependentluciferaseina dose-dependentmanner(Fig.6H).NHE1knockdownalsodecreasedthe expressionofbothNHE1andphospho-NF-𝜅Bp65inMDA-MB-231RR cells(Fig.6I).

InhibitionofNHE1inducestranscriptomicchangestoovercome radioresistanceinMDA-MB-231

WeusedRNAsequencingprofilestodeterminethetranscriptomic changesofMDA-MB-231(WT),MDA-MB-231RR(RR),andEIPA-treated MDA-MB-231RRcells(EIPA+RR)(Fig.7A).Weidentified490signifi- cantUp/DownDEGsforradioresistantcells(RRversusWT).TheseRR geneshadasignificant(p=1.5e-05)overlapwiththegenesfromRadia- tionGeneSigDB[36],whichcontainscuratedradiationresponse-related genesignatures(Fig.S2A).Theup-regulatedgenesareenrichedinpath- waysrelatedtocellularsenescence,immune,andextracellularmatrix (ECM),allofwhichhavebeenshowntocontributetoradioresistance [37,38]. Notably,EIPAsignificantlyreducedtheexpressionofsenes- cenceandECMpathwaygenes,implyingthattheradioresistantstatus wasalleviatedatthetranscriptomelevel(Fig.7B).

Toconfirmtranscriptionfactors(TFs)that maypotentiallybe in- volvedinradioresistanceandEIPA-inducedsensitization,weperformed enrichmentanalysisagainstaknown790TFdownstreamgenesetcol- lectedinHarmonizome[29],similartoconventionalpathway analy- sis(Fig.7C).NF-𝜅Banditsassociatedregulators,suchasLYL1,HTT, TRAF4, and IRF7 [39,40] were among the top 20 TFs. TGF𝛽 and epithelial-mesenchymaltransition (EMT)regulators werealso highly ranked,andtheseTFsareknowntoplayakeyroleinradioresistance [41].EIPAtreatmentsignificantlydownregulatedtheexpressionofthe majorityoftheseTFdownstreamgenesintheRRcells.Someofthese TFsmaybeinvolvedintheestablishmentofradioresistanceandEIPA- inducedsensitizationintheRRcells.

UsingSTRINGgenenetwork[30],weinvestigatedtheinteractions ofsignaturegenesfromthreekeyprocesses (NF-𝜅B,ECM,andsenes- cence)andfoundthatthesignaturegenesweretightlylinked(Fig.7D).

Theconnectivityofsignaturegenes(networkdensity=1.5e-02)was20 timesdenserthanthatoftherandomgenesinSTRINGnetwork(mean networkdensity=6.9e-03,p<0.001estimatedby1000timesofrandom samplednetworksofthesamesize).EIPAtreatmentfrequentlydown- regulated,or“flipped,” up-regulatedgenesinMDA-MB-231RRcells.For example,IL6andCXCL1weresignificantlyhigherinRRcells,which werereversedbyEIPA(Fig.7D).WealsoanalyzedEIPAdose-dependent changes inthese ’Flip genes’.Likethepathway analysis,’Flip genes’

weresignificantlyenrichedwiththethreekeyprocessesandothersig- naling pathways.Furthermore, most of these pathways weresignifi- cantlyenrichedwithgenesfromRadiationGeneSigDB(Fig.S2B).Over- all,inhibitingNHE1appearstofacilitatetranscriptomicchangesthat reversetheexpressionpatternofradioresistance-associatedgenesand pathways.

A. Son, S. Kang, S. Choi et al. Neoplasia 35 (2023) 100862

Fig.5. NHE1inhibitionincreasesradiation-inducedtumorgrowthdelayinvivo.(A)Treatmentschemeforionizingradiation(IR)andEIPAinanMDA-MB-231 tumorxenograftmodel.(B)MDA-MB-231xenografttumorgrowthcurvesinmicetreatedwithEIPAandIR.Thedatarepresentthemean±SD(n=5).(C)Changesin mousebodyweightduringtreatments.(D)RepresentativeTUNELstainingimagesoftheMDA-MB-231tumortissues.Tumorswereharvested54daysafterirradiation.

(E)TUNELpositivityquantification.(F)RepresentativeimmunohistochemistryimagesofNHE1andphospho-NF-𝜅Bp65inMDA-MB-231tumortissues.(G)NHE1 expressionquantification.(H)phospho-NF-𝜅Bp65expressionquantification.Thedatarepresentthemean±SD(n=9).^∗p<0.05;^∗∗p<0.01;^∗∗∗p<0.001.

Radioresistance-associatedpathwaysshowapositivecorrelationwithNHE1 expressioninTNBCpatients

Inorder tovalidate theclinical relevanceof NHE1forradioresis- tance,weanalyzedthetranscriptomeandthesurvivalofTNBCpatients extractedfromthebreastcancerdatasetofTCGA(Fig.S3A).Ofthe1222 BRCAsamples,239patientswereannotatedasTNBCaccordingtoFPKM ofERBB2,ESR1,andPGR(Materialsandmethods).These239TNBCpa- tientswereclassifiedasNES-highand–lowgroupsdependingontheex- pressionlevelofthethreekeyprocesseslistedinFig.7B.TheNES-high groupshowedasignificantlyhigherexpressionofNHE1(p=3.2e-04) (Fig.S3B).Furthermore,onlythepatientswhoreceivedradiotherapy

(n=123)showedasignificantdifferencebetweenNES-highand–low groupsinsurvival(Fig.S3C),wheretheNES-lowgroupshowedbetter overallsurvival,althoughonlymild(p=3.8e-02).Thesurvivaldiffer- encewasnotobservedinthepatientswithoutradiotherapy(n=81).

OurobservationsuggeststhathighNHE1expressionislikelytobeas- sociatedwithpoorsurvivalamongRT-treatedpatientspossiblythrough up-regulationoftheradioresistance-associatedprocesses.

Discussion

Radiationtherapyisoneofthemosteffectivestandardcancertreat- ments.Recentradiationtechniqueadvancements,suchasimage-guided

Fig.6.NHE1expressionandNF-𝜅BactivationareincreasedinradioresistanceMDA-MB-231cells.(A)SchemeforestablishingradioresistantMDA-MB-231cells (MDA-MB-231RR).Cellsthatsurvived40GyofX-rayswerepooledandsubjectedtoaclonogenicassay.(B)AclonogenicassayshowedthatMDA-MB-231RRcells wereradioresistantcomparedtotheirparentalcells.Thedatarepresentthemean±S.D(n=3).^∗p<0.05;^∗∗p<0.01.(C)MDA-MB-231RRcellshadincreasedpH_i comparedtoparentalcells.Thedatarepresentthemean±S.D(n=8).^∗∗∗p<0.001.(D)WesternblotanalysisshowedthatNHE1butnotMCT1wasinducedin MDA-MB-231RRcells.ThephosphorylationofNF-𝜅Bp65wasalsoincreased.𝛽-actinwasusedasaloadingcontrol.(E)MDA-MB-231RRcellsweremoresusceptible toEIPAthanparentalcells.(F)TreatmentwithEIPAre-sensitizedMDA-MB-231RRcellstoradiation.5μMEIPAwaspre-treated.(G)EIPAtreatmentreducedthe expressionofphospho-NF-𝜅Bp65inadose-dependentmanner.(H)EIPAinhibitedNF-𝜅B-dependentluciferaseactivity.Thedatarepresentthemean±S.D(n=4).

∗p<0.05;^∗∗∗p<0.001.(I)NHE1knockdownalsodecreasedtheexpressionofphospho-NF-𝜅Bp65.

radiotherapy andparticle beam therapy, have resulted in better tu- morcontrolwhileminimizingnormaltissuedamage.Nonetheless,tu- morcellsfrequentlydevelopradiationresistanceduringthetreatment, whichcanleadtorepopulationandcancerrelapse.Understandinghow tumorcellsacquireradiationresistanceisthereforecriticalforimprov- ingtreatmentoutcomes.AlthoughdysregulatedpH_iislinkedtocancer, itsroleinradioresistanceislargelyunknown.Weproposeinthispaper thattargetingNHE1,akeyregulatorofpH_i,isapreviouslyunrecognized buteffectivestrategyforovercomingradioresistance.

PreclinicalinvestigationofNHE1inhibitorshasbeenconductedas anti-cancerdrugsinpancreaticcancer[16],andfibrosarcoma[17]mod- els.Our findings consistentlydemonstrated that EIPA, anNHE1 in- hibitor,decreased theviabilityoftwocancercelllines.EIPAsignifi- cantlyenhancedradiation-inducedcelleradicationinMDA-MB-231and AsPC-1cells.Radiationalsoinhibitedthegrowthof3D-culturedMDA- MB-231andAsPC-1spheroids,whichwasfurtherinhibitedbytheaddi- tionofEIPA.EIPAdecreasedpH_iinaconcentration-dependentmanner.

AlowdoseofIRsuchas2GyraisedpH_i.NHE1activityisregulated bythedirectphosphorylation ofAKTandERK onitsC-terminal tail [18,42].Wespeculatedthatlow-doseirradiationmayactivateAKTor

ERK,resultinginanincreaseinpH_iwhichislikelytoprovideasurvival signal.Onthecontrary,highdosesabove6GydecreasedpH_i,despite increasedNHE1expression.Thismaybeduetomitochondrialdysfunc- tionandreactiveoxygenspecies(ROS)production,whichisknowntobe associatedwithapoptosis-mediatedintracellularacidification[5].The additionofEIPAtohigh-doseX-rayresultedinanevenlowerpH_i,po- tentiallyinhibitinggrowthinhibitionandcelldeath.Thespecificityof EIPAforNHE1inhibitionwassupportedbyNHE1knockdownexperi- ments.ThesefindingssuggestthatradiationmayaffectNHE1-regulated pH_ihomeostasis.Furtherinvestigationwillhelptounderstandhowra- diationregulatesNHE1activityorexpression.Directmeasurementof NHE1activityusingNH₄⁺prepulsecouldinformaboutsucheffects.

Ionizing radiation causesDNAdamage andcellcycle arrest, and theaccumulationofirreparableDNAdamageresultsincelldeath.De- layedexpressionof𝛾-H2AXandphosphorylatedCHK2indicatedthat EIPAattenuatedradiation-inducedDNAdamagerepair.Immunostain- ingof𝛾-H2AXrevealedmorefociinEIPA-treatedirradiatedcellsthan inDMSO-treatedirradiatedcells.Cellcycleanalysisfurtherconfirmed that EIPAincreased theradiation-induced G2/M arrest. Previous re- searchhasshownthatanNHE1-dependentincreaseinpH_iisrequired

A. Son, S. Kang, S. Choi et al. Neoplasia 35 (2023) 100862

Fig.7. Pharmacologicalinversionofradioresistance-associatedexpressionpatternsinMDA-MB-231cellsbyNHE1inhibition.(A)Anoverviewoftranscriptome analysesofradioresistantMDA-MB-231cellstreatedwithEIPA.GeneexpressionofMDA-MB-231RRandEIPA-treatedRRcellswasprofiled,andsignaturegenes forpathways,transcriptionfactors(TFs),andprotein-protein-interaction(PPI)networkswereidentified.(B)PathwaysenrichedinRRcells(RRversusWT)and EIPA-treatedRRcells(EIPAversusRR)relatedtoseveralkeyprocesses,includingsenescence,immune,andECM.Pathwaysthatareup-ordown-regulatedare coloredinredorblue,respectively.P-valuesarecalculatedbythehypergeometrictest,andthedottedlinesindicateasignificancecut-off (p<0.05,hypergeometric test).(C)TFanalysisforsignaturegenesthatareup-ordown-regulated.Thetop20mostsignificantTFsareorganizedintoknownfunctionalclasses.Thesignificance ofenrichmentwascalculatedusingahypergeometrictest,and790TFdownstreamgenesetswereobtainedfromHarmonizome.(D)Signaturegenenetworkinthe threekeyprocesses(NF-𝜅B,ECM,andsenescence).ThenetworksweretakenfromSTRINGwithaconfidencescore>0.4.Signaturegenesaredenotedbycircles, andadditionalTFsaredenotedbydiamonds(RELA,NFKB1,andTP53).TheinnercolorofanoderepresentstheexpressionchangeinRRvsWTgroup,andtheouter colorrepresentstheexpressionchangeintheEIPAversusRRgroup.

forG2/M entry[4], andNHE1 inhibitionreduced theexpression of componentsrequired for G2/M transition, such ascyclin B1, which mayexplaintheprolongedG2/MarrestafterirradiationandEIPA.In humanleukemiaHL-60cells,amiloride,aprototypeofEIPA,reduced theradiation-inducedG2block,resultingin delayingapoptosis[43],

whichsuggeststheeffectofamiloridederivativesonthecellcycleis cellcontext-dependent.

NHE1inhibitionincreasedradiation-inducedapoptosis.At50μM, EIPA alone significantly increased caspase 3/7 activity butwas no longerNHE1-specificasnoapoptoticinductionwasobservedinNHE1-

knockdowncells.Whencombinedwithradiation,EIPApre-treatment increasedcaspase3/7activityevenat20μM.EIPAplusX-rayincreased apoptoticmarkerscleavedPARPandBAKwhiledecreasing theanti- apoptoticproteinMCL-1.ThecombinationofEIPAandX-rayreduced thephosphorylatedformofNF-𝜅Bp65butnotthetotalprotein.EIPA orradiationaloneinhibitedthegrowthofMDA-MB-231tumorsin a xenografttumormousemodel.Co-administrationwithEIPAinhibited thegrowthofirradiatedtumorsevenmore.TheTUNELassayrevealed thatEIPA increasedapoptosisin tumortissuesexposed toradiation, whichwasconsistentwithourinvitrofindings.Furthermore,radiation increasedtheexpressionofNHE1andphosphorylatedNF-𝜅Bp65intu- mortissues, whichwassuppressedbyEIPAco-administration.These findingssuggestthatblockingNHE1mayhavearadiosensitizingeffect byincreasingapoptosis,whichmaybeinvolvedinthesuppressionof NF-𝜅Bsignaling.

NF-𝜅B is one of the transcription factorslinked to radiation re- sistance, including signals transduces and activator of transcription 3 (STAT3), nuclear factor (erythroid-derived-2)-like 2 (NRF2), and hypoxia-induciblefactor-1(HIF-1).Numerousgenesinvolvedindiverse cellularfunctions,suchascellproliferation,survival,andinflammation, areregulatedbyNF-𝜅B.Intermsofradioresistance,ionizingradiation activatesNF-𝜅BsignalingviaDNAdamage-mediatedATMactivation, cytokine-mediatedtumornecrosisfactorreceptor(TNF)signaling, or ROS-mediatedAKTactivation[44].ActivatedNF-𝜅Bincreasestheex- pressionofantioxidantgeneslikemitochondrialmanganesesuperoxide dismutase(MnSOD),anti-apoptoticgeneslikeBCL-2andBCL-xL,and EMT-relatedgeneslikeCOX-2andMMP1.Tobetterunderstandtheun- derlyingmechanismofNHE1-mediatedradioresistance,we createda radioresistantMDA-MB-231celllineandperformedawholetranscrip- tomicanalysis.RadioresistantcellshadahigherlevelofNHE1,pH_i,and phosphorylatedNF-𝜅Bp65thancontrolcells.Theradioresistantcells weremoresensitivetoEIPAthantheirparentalcells,whichcouldbe attributedtoadecreaseinNHE1andphosphorylatedNF-𝜅Bp65abun- dance.GeneexpressionprofilingconfirmedthattheNF-𝜅Bpathway, alongwithsenescenceandECM,wasoneof thecorepathways acti- vatedintheradioresistantgroup.Furthermore,comparativetranscrip- tomeanalysisonEIPA-treatedcellsdemonstratedthatblockingNHE1 reversedNF-𝜅B activationinradioresistant cells,implyinga rolefor NHE1andNF-𝜅Binadaptiveradioresistance.

LittleisknownabouthowNHE1orpH_imodulatesNF-𝜅Bsignaling inthepresenceofradiation.Innormaltissuessuchashumanumbili- calendothelialveincellsandgutepithelialcells,blockingNHE1inhib- itedtheNF-𝜅Bpathway,resultinginsuppressingtheinflammatoryre- sponse[45].NHE1inhibitionre-sensitizeschemoresistantMCF7cancer cellstodoxorubicinviaapoptosisactivationandNF-𝜅Bp65downreg- ulation[46].ThisisconsistentwithourfindingsthatNHE1inhibition abrogatedincreasedNHE1andNF-𝜅Bactivationintheirradiatedand radioresistantMDA-MB-231cells.AsidefromtheNF-𝜅Bpathway,our bioinformaticsanalysesrevealedthatTGF𝛽,EMT,andECMgeneswere upregulatedinradioresistancecells,whichwererelatedtometastatic potential.TGF𝛽inducesaSMAD4-dependentincreaseinNHE1andpH_i duringEMTinPanc-1pancreaticcancercells[47].EMT-driveninvasion necessitatesNHE1 transcriptionalupregulationviatheEMT-TF Zeb1 [48].Hypoxia-mediatedEMTandmigration/invasionofMDA-MB-231 cellswereinhibitedbyNHE1inhibition[49,50].Thus,inhibitingNHE1 couldreducemetastaticpotentialbypreventingradiation-inducedEMT.

Ourstudyhasseverallimitations.AlthoughNHE1isaprimarypH_i regulator,itremainsunclearwhethertheradiosensitizingeffectoftar- getingNHE1activityisdirectlydependentonpHi.Inhibitionofother pHmodifiers,suchasMCTs,shouldbetestedtodeterminewhetherthe resultsdependoneitherNHE1orpH_i.Recentoptogeneticstoolswillbe usefulfortestingthedirecteffectofpHichangesonradiationsensitivity [51].TheabsolutepH_ivalueofMDA-MB-231cellswasrelativelylower thanthatofmostcancercells,andinter-experimentalvariationwasalso observed.Nonetheless,thepHprobesusedwereadequateforcomparing relativepH_ichanges,andfutureresearchwilllookintootherpH-specific

probesforthispurpose.Anotherlimitationisthatintraperitonealadmin- istrationofEIPAintheMDA-MB-231xenograftmousemodelmakesit difficulttodeterminewhetherEIPAaffectstumorcellsinthetumormi- croenvironmentdirectlyorindirectly.AxenograftmodelusingNHE1 knockoutcellscouldbeamoreeffectivewaytodeterminethedirect roleofNHE1inradiosensitization.Finally,transcriptomicanalysisof theWT+EIPAgroupisimportanttoconfirmthattheTFdownstream genesdownregulatedbyEIPAarenotdependentonthedistinctcharac- teristicsofRRcells.

Giventhatadaptiveradioresistancemaybeamajorcauseoftumor recurrence,understandinghowtumorcells acquireradioresistanceis criticalfor overcomingradiotherapylocal failure.Ourfindings show that NHE1is adaptively upregulated duringradiation treatmentand highlyenrichedinthecellswithacquiredradioresistance.Inadditionto NF-𝜅B,NHE1inhibitionreversedradiation-inducedsenescence,suggest- ingthatNHE1inhibitionexertsasenolyticeffecttoeliminatesenescent cells.Theseradioresistance-associatedsignalingnetworks werefound tobe associatedwithhighNHE1expressioninTNBCpatientsamples fromtheTCGAdatabase,whichseemedtobeRT-specific.Furtherin- vestigationinmultiplepatientcohortswillberequiredtovalidatethe clinicalsignificanceofNHE1expressioninRTresponse.Inconclusion, ourintegratedapproachusinginvitro,invivo,andinsilicomethods suggeststhattargetingNHE1couldbeapromisingtherapeuticstrategy forimprovingRTefficacy.

Dataavailabilitystatement

Alldatageneratedandanalyzedduringthisresearcharepresentin thepaperand/orinthesupplementarymaterials.

DeclarationofCompetingInterest

Allauthorshavedeclarednoconflictofinterest.

CRediTauthorshipcontributionstatement

ArangSon:Validation, Formalanalysis, Investigation,Datacura- tion. SeoyeongKang: Validation, Investigation, Visualization. Suha Choi: Investigation,Formal analysis. Sung-WonShin: Investigation, Datacuration.YeeunKim:Investigation,Validation.WankyuKim:Re- sources,Writing– review&editing,Supervision,Fundingacquisition.

ChanghoonChoi:Conceptualization,Writing– originaldraft,Writing – review&editing,Projectadministration,Fundingacquisition.

Acknowledgments

This work was supported by grants from the National Re- search Foundation of Korea (NRF), funded by the Korean govern- ment(NRF-2021R1F1A1062064,NRF-2021M3H9A2098572andNRF- 2018R1D1A1B07042738).

Supplementarymaterials

Supplementarymaterialassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.neo.2022.100862.

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