1 plasma membrane ion channels and epithelial to ...413687/uq413687...5 98 ion channel activated by...

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Plasmamembraneionchannelsandepithelialtomesenchymaltransitionincancercells1

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ImanAzimi1,2,3,GregoryR.Monteith1,2,3*3

1TheSchoolofPharmacy,TheUniversityofQueensland,Brisbane,Queensland,Australia4

2MaterResearchInstitute,TheUniversityofQueensland,Brisbane,Queensland,Australia5

3TranslationalResearchInstitute,Brisbane,Queensland,Australia6

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*CorrespondenceshouldbeaddressedtoGRMonteith,Emailgregm@uq.edu.au8

Runningtitle:IonchannelsandEMTincancer9

10

Abstract11

Avarietyofstudieshavesuggestedthatepithelialtomesenchymaltransition(EMT)maybe12

importantintheprogressionofcancerinpatientsthroughmetastasisand/ortherapeuticresistance.13

AnumberofpathwayshavebeeninvestigatedinEMTincancercells.Recently,changesinplasma14

membraneionchannelexpressionasaconsequenceofEMThavebeenreported.Otherstudieshave15

identifiedspecificionchannelsabletoregulateaspectsofEMTinduction.Theutilityofplasma16

membraneionchannelsastargetsforpharmacologicalmodulationmakethemattractivefor17

therapeuticapproachestotargetEMT.Inthisreview,weprovideanoverviewofsomeofthekey18

plasmamembraneionchanneltypesandhighlightsomeofthestudiesthatarebeginningtodefine19

changesinplasmamembraneionchannelsasaconsequenceofEMTandalsotheirpossiblerolesin20

EMTinduction.21

22

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Introduction23

EpithelialtoMesenchymaltransition(EMT)referstotheprocesswherebyepithelialcellswhich24

typicallyexhibitfeaturessuchasstrongcelltocelladhesionandapical-basalpolarity,losethese25

propertiesandacquireotherssuchasgreatermotilityandaspindlelikemorphology(vanDenderen26

andThompson2013)(Thiery,etal.2009)(Fig.1).EMTisakeyeventindevelopmentalprocesses27

includingembryogenesiswhereitisassociatedwithimplantationandembryonicgastrulation(Kalluri28

andWeinberg2009).EMTisalsoafeatureofotheraspectsofnormalphysiologysuchaswound29

healingwhereithasanimportantroleintissueregeneration,andorganfibrosis(Kalluriand30

Weinberg2009).31

EMTincancer 32

Metastasisisthecauseofmortalityincancertypesthatoriginatefromorganswheresurgical33

resectionand/ortreatmentoftheprimarytumourareoftenfeasible(e.g.breastandprostate).34

Metastasisisahighlyregulatedprocesswherebycellsescapetheprimarytumour,enterthe35

circulatorysystemanddepositatametastaticsite(HanahanandWeinberg2011).Thereisclear36

coordinationofprocessesinmetastasisandthisisreflectedinthepropensityofdifferentcancer37

subtypestopreferentiallyformmetastaticlesionsinspecificsites.Thelossofcell-to-celladhesion,38

theacquisitionofmotility,theabilitytodegradethesurroundingextracellularmatrixandtosurvive39

stressessuchasthatinducedbyentryintothecirculationareallfeaturesthatarerequiredofcancer40

cellsduringmetastasis.Itisthereforenotsurprisingthatitisbelievedthatascellsleavetheprimary41

tumourtheymayundergoprocessessimilartoEMT(Heerboth,etal.2015).Theseincludethe42

expressionofthespecifictranscriptionfactorsSnailandTwist,expressionofmesenchymalmarkers43

suchasvimentinandN-Cadherin,andlossofepithelialmarkerssuchasE-cadherin(TsaiandYang44

2013).Indeed,theconsequencesofEMThavebeenreportedasincreasedmotilityandaremodelling45

ofcellularadhesion(Lamouille,etal.2014).EMTincancercellsisalsoassociatedwiththe46

acquisitionoftherapeuticresistance(SinghandSettleman2010).Althoughsomeveryrecentstudies47

3

indicatethatinsomecancersEMTmaybemoreimportantintheacquisitionoftherapeutic48

resistancethanmetastasis(Fischer,etal.2015;Zheng,etal.2015),understandingtheinductionof49

EMTandthepropertiesofthemesenchymalstatewouldclearlyhelpidentifynoveltherapeutic50

targets.51

AnumberoffactorsinthetumourmicroenvironmenthavebeenidentifiedasinducersofEMTin52

cancercells.Inbreastcancercells,growthfactorssuchasepidermalgrowthfactor(EGF),and53

hypoxiahavebeenshowntoinduceEMTinavarietyofinvitromodels,suchasMDA-MB-468breast54

cancercellsandZR-75-1breastcancercells(Davis,etal.2014a;Lester,etal.2007).Inprostate55

cancercells,EMTisinducedbyepidermalgrowthfactor(EGF)(Zhang,etal.2013b)andGrowthand56

differentiationfactor9(GDF-9)(Bokobza,etal.2011).Studiesinlungcancercellshave57

demonstratedthathypoxiainducesEMTthroughproteinkinaseA(PKA)activityinahypoxia-58

induciblefactor1-alpha(HIF1-α)dependentmanner(Shaikh,etal.2012).Avarietyofdrugable59

targetshavebeenidentifiedaspotentialmechanismstocontrolEMTinductionand/ortargetthe60

mesenchymalphenotypewhichisaconsequenceofEMT(Davis,etal.2014b).Oneclassofproteins61

thatarethetargetofexistingdrugsandmanydrugdevelopmentprogramsareionchannels.62

Plasmalemmalionchannelsinparticularareoftenamenabletopharmacologicalmodulationdueto63

theirextracellulardomains.Theavailabilityofselectiveinhibitorstospecificionchannelisoforms64

alsoallowschemogenomicandothermethodstodevelopnewtherapeutics.65

Ionchannelsasregulatorsofcellularprocesses66

Thepresenceofiongradientsacrosstheplasmamembraneisadefiningfeatureofmammaliancells.67

ThesodiumiongradientismaintainedbyNa+/K+-ATPasesthatactivelypumpNa+ionsfromthe68

cytoplasmtomaintainalowerintracellularfreeNa+levelcomparedtothoseoftheextracellular69

space(Castillo,etal.2015).Changesinthisgradientcanleadtorapidchangesinmembrane70

potentialanddriveactionpotentialsinexcitablecells.Similarly,changesincytosolicfreeCa2+71

([Ca2+]CYT)levelscanbemediatedbyactivationofCa2+permeableionchannelsandsuchchanges72

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haveimportantrolesinanarrayofcellularprocessesincludingfertilization,musclecontraction,73

hormonesecretion,genetranscriptionandcelldeath(Berridge,etal.2003).Thediversityof74

processesinfluencedbychangesinNa+,Ca2+andK+andotherionsthroughtheopeningofion75

channels,requiresthecelltoselectivelycontrolsuchchangesandthewaysuchchangesare76

decodedtoaltercellularprocesses.Hence,itisnotsurprisingthatthereareaplethoraofion77

channelsincells.Forexamplethereareover20genesthatencodeforjustonespecificclassofion78

channel-transientreceptorpotential(TRP)channelsinhumans.Thenextsectionprovidesan79

outlineofthegeneralpropertiesofionchannelsrelevanttothisreview.Wethenprovideaspecific80

overviewofstudiesthathaveidentifiedrolesofionchannelsinEMTinductionand/orremodelling.81

Plasmamembraneionchannels82

Thereareavarietyofionchannelswithdifferentpermeabilityandselectivityforcationsoranions.A83

comprehensivereviewofallionchannelsevenjustthoseoftheplasmamembraneiswellbeyond84

thescopeofthisreview.Hence,readersaredirectedtosourcesofcomprehensivelistsandreviewof85

ionchannelssuchastheIUPHAR/BPSguidetopharmacology(Southan,etal.2016),whichincludes86

otherchannelsnotdiscussedinthisreviewsuchasacid-sensing(proton-gated)ionchannels(ASICs)87

andsomeligandgatedCa2+channelssuchasionotropicglutamatereceptors.Arguably,themost88

extensivelystudiedplasmamembraneionchannelsarethosedepictedinFig.2–whichinclude89

calciumchannels,sodiumchannels,potassiumchannelsandchloridechannels.Examinationofeach90

ofthesechanneltypesprovidesinsightintotheirdiversity.Thesechannelscandifferdramaticallyin91

theirpropertiesfromionselectivitytotheirmechanismofactivation.92

ThediversityinionchannelpropertiesisclearintheplasmamembraneCa2+channelspresentedin93

Fig.2–Orai,TRP,P2XandvoltagegatedCa2+channels(VGCC).Theseclasseshavecleardifferences94

intheirmechanismofactivation.ForexampletheOrai1proteinispartofacomplexwherebyCa2+95

influxisactivatedbythedepletionofendoplasmicreticulumCa2+stores(Azimi,etal.2014).In96

contrast,TRPchannelshavebeendescribedassensors,asexemplifiedbyTRPV1aCa2+permeable97

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ionchannelactivatedbyheatandthehotchillicomponent,capsaicin(Azimietal.2014).Other98

ligandgatedcalciumchannelsincludeionotropicglutamatereceptorsandalsoP2Xchannelsthatare99

activatedbysomenucleosides(e.g.ATP)whereasVGCCsareactivatedbychangesinmembrane100

potential(Azimietal.2014).EvenwithinclassesofCa2+channelsthereisgreatdiversityofactivators101

(e.g.TRPV1isactivatedbycapsaicinwhereasTRPM8isactivatedbymenthol)andionselectivity(e.g.102

TRPV6ishighlyselectiveforCa2+ionswhereasTRPV1isalsopermeabletoNa+ions)(Azimietal.103

2014).TheremodellingofCa2+channelexpressionhasbeendefinedinsomecancersandsomehave104

beenidentifiedaspotentialtherapeutictargetsinsomecancersubtypesasreviewedelsewhere105

(Azimietal.2014;Stewart,etal.2015).Indeed,SOR-C13,aTRPV6inhibitorhasbeenrecently106

assessedinclinicaltrialsofovariancancer(www.clinicaltrials.gov,NCT01578564).107

AlthoughtheassociationbetweenNa+influxandactionpotentialshasseenafocusonNa+channel108

inneuroscienceandcardiovascularresearch,Na+channelsareinfactexpressedinavarietyofcell109

types.Forexamplevoltagegatedsodiumchannels(VGSC)areexpressedinexcitablecellsincluding110

neuronsandmusclecells,wheretheyareresponsibleforactionpotentialandconduction(Southan111

etal.2016);NALCNhasbeendescribedasasodiumleakchannelwhichregulatestheresting112

membranepotentialandexcitabilityinneurons(Cochet-Bissuel,etal.2014);andepithelialsodium113

channels(ENaC)playpivotalrolesintheregulationofextracellularfluid(ECF)volumeandblood114

pressureinkidneytubules(HanukogluandHanukoglu2016).Potassiumchannelsareequallyas115

complexanddiverseandincludethosethatarevoltagegated(VGKC),thosethataretwo-pore116

domain(K2P),thosethatplayrolesinCa2+-activatedK+transport(KCachannels)andInwardly117

rectifyingK+(IRK)channels(Hibino,etal.2010).118

Chloridechannelsincludechannelsthatwhendefectiveduetohereditarymutationcanalterthe119

fluidtransportinepithelialcellsresultingincysticfibrosis(CysticFibrosisTransmembrane120

conductanceRegulator(CFTR)),channelsactivatedbyintracellularCa2+(CaCC),thosewhichare121

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ligandactivated(LGCC),orvolumeregulated(VRAC)orthechloridechannelsuperfamily(CIC)122

(Southanetal.2016).123

Theoutlineofplasmalemmalionchannelspresentedabovehighlightedthediversityofionchannels124

andtheirrolesinmammaliancells.Asdiscussedbelow,someoftheseionchannelshaverecently125

beenshowntoberemodelledasaconsequenceofEMTincancercellsorplayrolesintheinduction126

ofEMTmarkersinducedbysomestimuli.127

PlasmamembraneionchannelsandEMTincancercells128

ThisreviewisfocusedontheremodellingandinsomecasesrolesofionchannelsinEMTincancer129

cells.ItshouldbenotedthatotherstudieshaveinvestigatedionchannelsinEMTinthecontextof130

otherEMTrelevantprocessesmanyofwhichintersectwithdiseasestatessuchasairway131

remodelling(Arthuretal.,2015)andrenalfibrosis(Maietal.,2016).132

133

Theverydifferentpropertiesofcancercellssuchastheacquisitionoftherapeuticresistanceandthe134

majorchangesintheexpressionofspecificproteins(e.g.vimentin)andtranscriptionfactors(e.g.135

twistandSnail)asaconsequenceofEMTmeansthatchangesinioninfluxshouldnothavebeen136

surprising.ThechangeinphenotypeofcancercellsthathaveundergoneEMTandtheveryspecific137

rolesofspecificionchannelsindifferentcelltypessuggeststhatthemesenchymalphenotypewill138

exploitdifferentionchannelstoachievedifferentcellularfunctions.Inthesectionbelowwewill139

provideanoverviewofstudiesthathavenowshownsuchchangesandinsomecasesimplicated140

specificionchannelsinEMTinduction.ManyofthesestudiesaresummarisedinTable1.141

SodiumchannelsandEMTincancercells142

Hypotheseshavebeenproposedandanintellectualcasemadeforthepotentialofvoltage-gated143

sodiumchannelstoregulateEMTinductionincancercells(ErenandOyan2014;Eren,etal.2015).144

Therepositioningofclinicallyusedvoltage-gatedsodiumchannelblockerstoattenuatemetastatic145

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progressionand/orchemotherapyresistancethroughinhibitionofEMTinductionhasalsobeen146

highlighted(Erenetal.2015).However,thisareahasyettobefullyassessedexperimentallywith147

modelsofEMTincancercells,andthisrepresentsanopportunityforfutureresearch.148

PotassiumchannelsandEMTincancercells149

TheassociationbetweenchangesinthepotassiumgradientandEMTwassuggestedinearlystudies150

ofpotassiumchlorideco-transporter3(KCC3)(Hsu,etal.2007).KCC3isnotanionchannel,butits151

abilitytocotransportK+andCl-ionsmakesitanimportantregulatorofthefluxoftheseionsacross152

theplasmamembranesofmanycelltypeswhereitcanplayanimportantroleintheregulationof153

cellvolume(Hsuetal.2007;Kahle,etal.2015).ForcedoverexpressionofKCC3incervicalcancer154

SiHacellsisassociatedwiththeadoptionofamoremesenchymal-likemorphology,thedown155

regulationoftheepithelialmarkerE-cadherinandtheupregulationofthemesenchymalmarker156

vimentin(Hsuetal.2007).SubsequenttothesestudiesanassociationwiththeEAG1potassium157

channelandEMTinlungcancercellshasbeenimplicated,becauseofanincreaseinEag1mRNA158

levelsinA549lungcancercellstreatedwithtransforminggrowthfactorbeta1(TGFβ1),anEMT159

inducerinthismodel(Restrepo-Angulo,etal.2011).Incontextofcolorectalcancer,studiesof160

phosphataseofregeneratingliver-3(PRL-3)inducedEMTinLoVocells(acoloncancercellline),has161

shownthatapharmacologicalinhibitoroftheCa2+activatedpotassiumchannelKCNN4-TRAM-34,162

supressesthemesenchymalmarkersvimentinandSnail,andincreasestheexpressionofthe163

epithelialmarkerE-cadherin(Lai,etal.2013).AlthoughtheconcentrationsofTRAM-34usedmay164

haveinhibitedotherionchannels,siRNAtoKCNN4phenocopiedtheeffectsofTRAM-34(Laietal.165

2013).Moreover,KCNN4expressionwaspositivelycorrelatedwithtumourstageinclinicalcohortof166

86patientcolorectaltumoursamples(Laietal.2013).Veryrecentstudieshavenowshownthat167

silencingofKCNN4inMDA-MB-231(abreastcancercelllinewithfeaturesofthemesenchymal168

phenotype)appearedtoreducetheexpressionofthemesenchymalmarkersvimentinandSnail1169

(Zhang,etal.2016).170

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Collectively,thestudiesdescribedabovearebeginningtodefineassociationsbetweenspecific171

potassiumchannelsandEMTincancercells.Furtherstudiesofotherpotassiumchannelsinthe172

contextofchangesinexpressionasaconsequenceofEMTaswellastheinductionofEMTand/or173

maintenanceofthemesenchymalphenotypenowseemappropriate.GiventhediversityofEMT174

modelsincancercellsandthevarietyofinducersofEMT,itisalsoimportantthattherolesof175

specificpotassiumchannelsbeinvestigatedacrossavarietyofmodelsandinducersofEMT.176

ChloridechannelsandEMTincancercells177

Anincreasingnumberofstudieshaveidentifiedtheremodellingofexpressionofchloridechannel178

componentsasaconsequenceofEMTincancercells.Examplesofsuchremodellingincludeisoforms179

ofchloridechannelaccessoryproteins,namelyCLCA2andCLCA4.CLCA2mRNAlevelsarereducedin180

breastcancercelllinesassociatedwiththemesenchymalphenotype(e.g.MDA-MB-231andBT549)181

comparedtothoseoftenenrichedinepithelialmarkers(e.g.MCF-7).Indeed,expressionoftheEMT182

transcriptionfactorSnailsupressesCLCA2proteininthehumanbreastcelllineMCF10A,andCLCA2183

levelsarereducedinsubpopulationsofcellsfromthehumanmammaryepithelial(HMLE)cellline184

thatareenrichedinmesenchymalmarkers(Walia,etal.2012).Moreover,CLCA2levelsarereduced185

duringEMTinducedbyTGFβ(Yu,etal.2013).Similarly,reducedlevelsoftherelatedisoformCLCA4186

isafeatureofsubpopulationsofcellsfromtheHMLEcelllinethatareenrichedinmesenchymal187

markersandaconsequenceofTGFβ-inducedEMT(Yuetal.2013).ConsistentwiththelossofCLCA2188

andCLCA4inthemesenchymalphenotype,lowlevelsofCLCA2andCLCA4appearlikelytobe189

associatedwithanincreasedincidenceofmetastasis(asassessedthroughmetastasisorrelapsefree190

survival)usingspecificcohortsofbreastcancerpatients(Waliaetal.2012;Yuetal.2013).In191

additiontotheirremodellingasaconsequenceofEMT,CLCA2andCLCA4havealsobeenimplicated192

intheregulationofthetransitionofbreastcancercellstowardsamoremesenchymalstate.193

KnockdownofCLCA2orCLCA4issufficientinHMLEcellstoinducetheexpressionofthe194

mesenchymalmarkervimentinandsupresstheepithelialmarkerE-Cadherin(Waliaetal.2012;Yuet195

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al.2013).InthecaseofCLCA2,theregulationofEMTmayatleastinpartbethroughinteractions196

withthecelljunctionalproteinEVA1(Ramena,etal.2016).Futurestudiesarenowrequiredto197

definetherelativeimportanceinchangesinchloridefluxintheseevents,andtheabilityoftheloss198

ofCLCA2orCLCA4toinduceamesenchymalphenotypeinothermodelsofEMT,includingthosenot199

ofbreastcancerorigin.200

Breastcancercellshavealsobeenthefocusofinvestigatorsexploringtherelationshipbetween201

CFTRandEMT.TheEMTinducerTGFβ1causesadownregulationofCFTRinMCF-7cells,whichis202

alsoassociatedwithadownregulationoftheepithelialmarkerE-cadherin(Zhang,etal.2013a).A203

functionalroleforCFTRinEMTinductionissuggestedbytheabilityofCFTRsilencingtoinducethe204

expressionofavarietyofmesenchymalmarkersinMCF-7breastcancercells.Thisproposedfunction205

ofCFTRisfurthersupportedbytheabilityofCFTRoverexpressioninmesenchymal-likeMDA-MB-206

231breastcancercellstosuppresstheexpressionofvimentin(amesenchymalmarker)andinduce207

theexpressionofE-cadherin(anepithelialmarker)(Zhangetal.2013a).Aswouldbepredicted208

basedontheseresults,reducedlevelsofCFTRareassociatedwithpoorprognosisinbreastcancer209

patients(Zhangetal.2013a).Morerecentstudieshavebeguntoexplorechloridechannelsinthe210

contextofEMTinothercancertypes,suchassquamouscellcarcinomasoftheheadandneck211

(Shiwarski,etal.2014).TMEM16A(alsoknownasANO1),isoneofareportedsubset(termed212

Anoctamins)ofcalciumactivatedchloridechannels(Kunzelmann,etal.2011).LevelsofTMEM16A213

arereducedincancercellsinmetastaticlymphnodescomparedtothoseoftheprimarytumourin214

squamouscellcarcinomasoftheheadandneck(Shiwarskietal.2014).TMEM16Aseemstobe215

morethanapotentialmarkerofEMT,sincesilencingofTMEM16AinT24cells(ahumanbladder216

carcinomacellline),producesamesenchymal-likephenotype(spindlemorphology,lowerE-217

cadherin,increasedSnail)andoverexpressionofTMEM16Aproducesanepithelial-likephenotype218

(roundedpackedmorphology,increasedE-cadherin,reducedvimentinandfibronectin)(Shiwarski219

etal.2014).220

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Theworkdescribedabove,performedbyavarietyofinvestigatorsusinganarrayofmodelsand221

approacheshasnowhelpeddefinearemodellingofspecificchloridechannels(orcomponents)in222

EMTandaroleforthesesamechannelsintheinductionofEMTand/orthemaintenanceofthe223

epithelial-likephenotype.224

CalciumchannelsandEMTincancercells225

Thecalciumsignalhasbeenidentifiedasorcouldbespeculatedtobeapotentialmechanismby226

whichatleastsomeoftheaforementionedionchannelsmayimmediatetheireffectsonEMT.For227

examplethemechanismbywhichKCNN4mayregulateEMTincoloncancercellshasbeenlinkedto228

effectsoncalciumsignalling(Laietal.2013).Indeed,globalchelationofintracellularfreeCa2+that229

attenuatesincreasesincytosolicfreeCa2+,suppressesbothEGFandhypoxiainducedincreasesinthe230

mesenchymalmarkersvimentin,N-cadherinandCD44(Davisetal.2014a).Similarfindingshave231

nowbeenreportedinHuh7andHepG2hepaticcancercelllinesforEMTinducedbydoxorubicin232

(Wen,etal.2016).Itisalsonowclearthatamajorremodellingincalciumsignallingandthe233

expressionofspecificcalciumpermeableionchannelsisafeatureofEMTandsomecalcium234

permeableionchannelsareimportantintheinductionofexpressionofsomeproteinsassociated235

withthemesenchymalphenotype.236

AlterationsintheresponsestoATP,abletoactivateG-proteincoupledpurinergicreceptors(P2Y237

family)andligandgatedCa2+channels(P2Xfamily)isafeatureofbothEGFandhypoxiainducedEMT238

inMDA-MB-468breastcancercells(Azimi,etal.2015;Davis,etal.2011).EMTinducedbyhypoxia239

andEGFisassociatedwiththeattenuationofpeak[Ca2+]CYTandthesustainedphaseofCa2+influx240

inducedbyATP.EMTisalsoassociatedwithareductioninthesensitivitytoATPwithanincreasethe241

EC50(Azimietal.2015;Davisetal.2011).Suchchangesinthemesenchymalphenotypemaybean242

adaptionofbreastcancercellstothehighATPconcentrationsinsometumourmicroenvironments.243

However,despitethisconsistentchangeinATP-mediatedCa2+signalling,thenatureofthe244

remodellingofP2XreceptorsseemsverydifferentastheupregulationofP2X5mRNAisafeatureof245

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EGFbutnothypoxiaassociatedATP(Azimietal.2015;Davisetal.2011).Theattenuationofstore246

operatedCa2+entry(SOCE)andbasalCa2+influxisalsoafeatureofEGFinducedEMTinMDA-MB-247

468(Davis,etal.2012),however,assessmentofsuchchangeswithhypoxiainducedEMThasnot248

beenreported.SuchstudiesarecriticalgiventhatinMCF-7cells,theEMTinducerTGF-β1hasbeen249

reportedtobeassociatedwithenhancementofstoreoperatedCa2+entry(Hu,etal.2011).250

InadditiontoaremodellingofCa2+influxand/ortheexpressionofsomeCa2+permeableion251

channelsinEMTincancercells,specificcalciumpermeableionchannelshavealsobeenidentifiedas252

regulatorsoftheinductionofatleastsomehallmarksofEMT.AfocusedsiRNAscreenidentified253

TRPM7asaregulatorofEGF-inducedexpressionofthemesenchymalmarkervimentininMDA-MB-254

468breastcancercells(Davisetal.2014a).ApharmacologicalinhibitorofTRPM7replicatedthe255

consequencesofTRPM7silencingonEGFinducedvimentinexpression.Theseeffectswerenotdue256

togeneralinhibitionofEGFreceptor(EGFR)signallingsinceEGF-mediatedEGFRandAKT257

phosphorylationwereunaffectedbyTRPM7silencing,however,EGF-mediatedSTAT3andERK1/2258

phosphorylationweresignificantlyreduced(Davisetal.2014a).AlthoughaCa2+permeableion259

channel,theimportanceofTRPM7inMg2+homeostasisanditsabilitytofunctionasanatypical260

alphakinase(Paravicini,etal.2012)requirefurtherattentionintothenatureofitscontributionto261

EMTinsomecancermodels.SilencingofthecoldsensorTRPM8increasestheexpressionofthe262

epithelialmarkerE-cadherininmesenchymal-likeMDA-MB-231cellsandreduceslevelsofthe263

mesenchymalmarkervimentin(Liu,etal.2014).ConsistentwitharoleforTRPM8inthe264

maintenanceand/orinductionofthemesenchymalphenotypeinbreastcancercells,overexpression265

ofTRPM8inthemoreepitheliallikeMCF-7celllineleadstoEMTinductionasindicatedbydown266

regulationofE-cadherinandinductionofvimentin(Liuetal.2014).InHuh7andHepG2hepatic267

cancercells,TRPC6silencingattenuateschangesintheexpressionofE-cadherininducedby268

doxorubicinsuggestingthatintheabilityofTRPC6silencingtoincreasesensitivitytodoxorubicin269

througheffectsofresistancepathwaysmaybedueatleastinparttoeffectsonsomeaspectsofEMT270

induction(Wenetal.2016).271

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Hence,studiesofcalciumsignallingandCa2+permeableionchannelsinEMTfromavarietyofgroups272

usinganarrayofEMTinducersandmodelshavehelpeddefineacriticalrolefortheCa2+signalin273

EMTincancercells.274

275

Conclusion276

Anincreasingnumberofstudieshavereportedtheremodellingofplasmamembraneionchannel277

expressionasacharacterizingfeatureofEMTincancercells.Theidentificationoftheroleofspecific278

ionchannelsintheinductionofEMTand/orthemaintenanceofaspectsoftheepithelialor279

mesenchymal-likephenotypeincancercellssuggestthatsomeionchannelsmaybetherapeutic280

targetstocontrolEMTandhencediseaseprogression(e.g.therapeuticresistance).However,itis281

likelythatdifferentEMTinducersmayengagedifferentionchannelstoregulatethepropertiesof282

themesenchymalphenotypeand/orEMTinductionitself.Thisissueandthestudyofthe283

intersectionbetweensexhormonesandreceptorsthatregulateEMT(Jeon,etal.2016;Kong,etal.284

2015;Zuo,etal.2010;Sun,etal.2014;vanderHorst,etal.2012)andionchannelswhich285

themselvesintersectwithsexhormonepathways(Asuthkar,etal.2015;Hao,etal.2015;286

Mahmoodzadeh,etal.2016)areareasforfutureresearch.Whichionchannelstopursuefor287

therapeutictargetingrequirescarefulconsideration,anddecidingfactorswillincludetheexpression288

oftargetsinothercelltypesandthelikelyadversesystemiceffectsofchannelinhibitors.However,289

thesuccessfuluseofionchannelinhibitorsforconditionsasdiverseascardiovasculardiseaseto290

paindemonstratedtheneedtocontinueresearchinthisarea.291

292

DeclarationofInterest293

GRMisassociatedwithQUEOncologyInc.294

295

13

Funding296

TheresearchwassupportedbytheNationalHealthandMedicalResearchCouncil(NHMRC;project297

grant1079672).GRMwassupportedbytheMaterFoundation.TheTranslationalResearchInstitute298

issupportedbyagrantfromtheAustralianGovernment.299

300

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