1 plasma membrane ion channels and epithelial to ...413687/uq413687...5 98 ion channel activated by...
TRANSCRIPT
1
Plasmamembraneionchannelsandepithelialtomesenchymaltransitionincancercells1
2
ImanAzimi1,2,3,GregoryR.Monteith1,2,3*3
1TheSchoolofPharmacy,TheUniversityofQueensland,Brisbane,Queensland,Australia4
2MaterResearchInstitute,TheUniversityofQueensland,Brisbane,Queensland,Australia5
3TranslationalResearchInstitute,Brisbane,Queensland,Australia6
7
*CorrespondenceshouldbeaddressedtoGRMonteith,[email protected]
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
2
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
4
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
5
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
6
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
7
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
8
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
9
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
10
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
11
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
12
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
References301
ArthurGK,DuffySM,RoachKM,HirstRA,ShikotraA,GaillardEA&BraddingP2015K(Ca)3.1K+302
ChannelExpressionandFunctioninHumanBronchialEpithelialCells.PLoSONE10e0145259.303
AsuthkarS,ElustondoPA,DemirkhanyanL,SunX,BaskaranP,VelpulaKK,ThyagarajanB,PavlovEV304
&ZakharianE2015TheTRPM8ProteinIsaTestosteroneReceptorI.Biochermicalevidencefor305
directTRPM8-testoesteroneintersactions.JournalofBiologicalChemistry2902659-2669.306
AzimiI,BeilbyH,DavisFM,MarcialDL,KennyPA,ThompsonEW,Roberts-ThomsonSJ&Monteith307
GR2015Alteredpurinergicreceptor-Casignalingassociatedwithhypoxia-inducedepithelial-308
mesenchymaltransitioninbreastcancercells.MolecularOncology10166–178.309
AzimiI,Roberts-ThomsonSJ&MonteithGR2014Calciuminfluxpathwaysinbreastcancer:310
opportunitiesforpharmacologicalintervention.BritishJournalofPharmacology171945-960.311
BerridgeMJ,BootmanMD&RoderickHL2003Calciumsignalling:dynamics,homeostasisand312
remodelling.NatureReviews.MolecularCellBiology4517-529.313
BokobzaSM,YeL,KynastonH&JiangWG2011Growthanddifferentiationfactor9(GDF-9)induces314
epithelial-mesenchymaltransitioninprostatecancercells.MolecularandCellularBiochemistry349315
33-40.316
CastilloJP,RuiH,BasilioD,DasA,RouxB,LatorreR,BezanillaF&HolmgrenM2015Mechanismof317
potassiumionuptakebytheNa+/K+-ATPase.NatureCommunications67622.318
14
Cochet-BissuelM,LoryP&MonteilA2014Thesodiumleakchannel,NALCN,inhealthanddisease.319
FrontiersinCellularNeuroscience8132.320
DavisFM,AzimiI,FavilleRA,PetersAA,JalinkK,PutneyJrJW,GoodhillGJ,ThompsonEW,Roberts-321
ThomsonSJ&MonteithGR2014aInductionofepithelial-mesenchymaltransition(EMT)inbreast322
cancercellsiscalciumsignaldependent.Oncogene332307-2316.323
DavisFM,KennyPA,SooET,vanDenderenBJ,ThompsonEW,CabotPJ,ParatMO,Roberts-Thomson324
SJ&MonteithGR2011Remodelingofpurinergicreceptor-mediatedCa2+signalingasaconsequence325
ofEGF-inducedepithelial-mesenchymaltransitioninbreastcancercells.PLoSONE6e23464.326
DavisFM,PetersAA,GriceDM,CabotPJ,ParatMO,Roberts-ThomsonSJ&MonteithGR2012Non-327
stimulated,agonist-stimulatedandstore-operatedCa2+influxinMDA-MB-468breastcancercells328
andtheeffectofEGF-inducedEMToncalciumentry.PLoSONE7e36923.329
DavisFM,StewartTA,ThompsonEW&MonteithGR2014bTargetingEMTincancer:opportunities330
forpharmacologicalintervention.TrendsinPharmacologicalSciences35479-488.331
ErenOO&OyanB2014Voltage-gatedsodiumchannelblockadeforinhibitionofEMT.Trendsin332
PharmacologicalSciences35621-621.333
ErenOO,OzturkMA,SonmezOU&OyanB2015Voltage-gatedsodiumchannelblockerscan334
augmenttheefficacyofchemotherapeuticsbytheirinhibitoryeffectonepithelial-mesenchymal335
transition.MedicalHypotheses8411-13.336
FischerKR,DurransA,LeeS,ShengJT,LiFH,WongSTC,ChoiHJ,ElRayesT,RyuSH,TroegerJ,etal.337
2015Epithelial-to-mesenchymaltransitionisnotrequiredforlungmetastasisbutcontributesto338
chemoresistance.Nature527472-476.339
HanahanD&WeinbergRA2011HallmarksofCancer:TheNextGeneration.Cell144646-674.340
HanukogluI&HanukogluA2016Epithelialsodiumchannel(ENaC)family:Phylogeny,structure-341
function,tissuedistribution,andassociatedinheriteddiseases.Gene57995-132.342
15
HaoJ,BaoX,JinB,WangX,MaoZ,LiX,WeiL,ShenD&WangJL2015Ca2+channelsubunitalpha343
1Dpromotesproliferationandmigrationofendometrialcancercellsmediatedby17beta-estradiol344
viatheGprotein-coupledestrogenreceptor.FASEBJournal292883-2893.345
HeerbothS,HousmanG,LearyM,LongacreM,BylerS,LapinskaK,WillbanksA&SarkarS2015EMT346
andtumormetastasis.ClinicalandTranslationalMedicine46.347
HibinoH,InanobeA,FurutaniK,MurakamiS,FindlayI&KurachiY2010InwardlyRectifying348
PotassiumChannels:TheirStructure,Function,andPhysiologicalRoles.PhysiologicalReviews90349
291-366.350
HsuYM,ChenYF,ChouCY,TangMJ,ChenJH,WilkinsRJ,ElloryJC&ShenMR2007KCI351
cotransporter-3down-regulatesE-cadherin/beta-catenincomplextopromoteepithelial-352
mesenchymaltransition.CancerResearch6711064-11073.353
HuJJ,QinKH,ZhangY,GongJB,LiN,LvD,XiangR&TanXY2011Downregulationoftranscription354
factorOct4inducesanepithelial-to-mesenchymaltransitionviaenhancementofCa2+influxin355
breastcancercells.BiochemicalandBiophysicalResearchCommunications411786-791.356
JeonSY,HwangKA&ChoiKC2016Effectofsteroidhormones,estrogenandprogesterone,on357
epithelialmesenchymaltransitioninovariancancerdevelopment.JournalofSteroidBiochemistry358
andMolecularBiology1581-8.359
KahleKT,KhannaAR,AlperSL,AdragnaNC,LaufPK,SunDD&DelpireE2015K-Clcotransporters,360
cellvolumehomeostasis,andneurologicaldisease.TrendsinMolecularMedicine21513-523.361
KalluriR&WeinbergRA2009Thebasicsofepithelial-mesenchymaltransition.JournalofClinical362
Investigation1191420-1428.363
KongDJ,SethiS,LiYW,ChenW,SakrWA,HeathE&SarkarFH2015AndrogenReceptorSplice364
VariantsContributetoProstateCancerAggressivenessThroughInductionofEMTandExpressionof365
StemCellMarkerGenes.Prostate75161-174.366
16
KunzelmannK,TianYM,MartinsJR,FariaD,KongsupholP,OusingsawatJ,ThevenodF,RoussaE,367
RockJ&SchreiberR2011Anoctamins.PflugersArchiv-EuropeanJournalofPhysiology462195-208.368
LaiW,LiuL,ZengYJ,WuH,XuHY,ChenS&ChuZH2013KCNN4ChannelsparticipateintheEMT369
inducedbyPRL-3incolorectalcancer.MedicalOncology30566.370
LamouilleS,XuJ&DerynckR2014Molecularmechanismsofepithelial-mesenchymaltransition.371
NatureReviewsMolecularCellBiology15178-196.372
LesterRD,JoM,MontelV,TakimotoS&GoniasSL2007uPARinducesepithelial-mesenchymal373
transitioninhypoxicbreastcancercells.JournalofCellBiology178425-436.374
LiuJX,ChenYZ,ShuaiS,DingDP,LiR&LuoRC2014TRPM8promotesaggressivenessofbreast375
cancercellsbyregulatingEMTviaactivatingAKT/GSK-3betapathway.TumorBiology358969-8977.376
MahmoodzadehS,HaaseH,SporbertA,RharassT,PanakovaD&MoranoI2016Nuclear377
translocationofthecardiacL-typecalciumchannelC-terminusisregulatedbysexand17beta-378
estradiol.JournalofMolecularandCellularCardiology97226-234.379
MaiX,ShangJ,LiangS,YuB,YuanJ,LinY,LuoR,ZhangF,LiuY,LvX,etal.2016BlockadeofOrai1380
Store-OperatedCalciumEntryProtectsagainstRenalFibrosis.JournaloftheAmericanSocietyof381
Nephrology.382
ParaviciniTM,ChubanovV&GudermannT2012TRPM7:Auniquechannelinvolvedinmagnesium383
homeostasis.InternationalJournalofBiochemistry&CellBiology441381-1384.384
RamenaG,YinYF,YuY,WallaV&ElbleRC2016CLCA2InteractorEVA1IsRequiredforMammary385
EpithelialCellDifferentiation.PLoSONE11e0147489.386
Restrepo-AnguloI,Sanchez-TorresC&CamachoJ2011HumanEAG1PotassiumChannelsinthe387
Epithelial-to-MesenchymalTransitioninLungCancerCells.AnticancerResearch311265-1270.388
17
ShaikhD,ZhouQY,ChenTJ,IbeJCF,RajJU&ZhouGF2012cAMP-dependentproteinkinaseis389
essentialforhypoxia-mediatedepithelial-mesenchymaltransition,migration,andinvasioninlung390
cancercells.CellularSignalling242396-2406.391
ShiwarskiDJ,ShaoCB,BillA,KimJ,XiaoD,BertrandCA,SeethalaRS,SanoD,MyersJN,HaP,etal.392
2014To"Grow"or"Go":TMEM16AExpressionasaSwitchbetweenTumorGrowthandMetastasis393
inSCCHN.ClinicalCancerResearch204673-4688.394
SinghA&SettlemanJ2010EMT,cancerstemcellsanddrugresistance:anemergingaxisofevilin395
thewaroncancer.Oncogene294741-4751.396
SouthanC,SharmanJL,BensonHE,FaccendaE,PawsonAJ,AlexanderSPH,BunemanOP,Davenport397
AP,McGrathJC,PetersJA,etal.2016YTheIUPHAR/BPSGuidetoPHARMACOLOGYin2016:towards398
curatedquantitativeinteractionsbetween1300proteintargetsand6000ligands.NucleicAcids399
Research44D1054-D1068.400
StewartTA,YapaKTDS&MonteithGR2015Alteredcalciumsignalingincancercells.BiochimicaEt401
BiophysicaActa-Biomembranes18482502-2511.402
SunY,WangYS,FanC,GaoP,WangXW,WeiGW&WeiJM2014Estrogenpromotesstemnessand403
invasivenessofER-positivebreastcancercellsthroughGli1activation.MolecularCancer13137.404
ThieryJP,AcloqueH,HuangRYJ&NietoMA2009Epithelial-MesenchymalTransitionsin405
DevelopmentandDisease.Cell139871-890.406
TsaiJH&YangJ2013Epithelial-mesenchymalplasticityincarcinomametastasis.Genes&407
Development272192-2206.408
vanDenderenBJW&ThompsonEW2013CANCERThetoandfrooftumourspread.Nature493487-409
488.410
18
vanderHorstPH,WangYY,VandenputI,KuhneLC,EwingPC,vanIJckenWFJ,vanderZeeM,Amant411
F,BurgerCW&BlokLJ2012ProgesteroneInhibitsEpithelial-to-MesenchymalTransitionin412
EndometrialCancer.PLoSONE7e30840.413
WaliaV,YuY,CaoD,SunM,McLeanJR,HollierBG,ChengJ,ManiSA,RaoK,PremkumarL,etal.414
2012LossofbreastepithelialmarkerhCLCA2promotesepithelial-to-mesenchymaltransitionand415
indicateshigherriskofmetastasis.Oncogene312237-2246.416
WenL,LiangC,ChenEJ,ChenW,LiangF,ZhiX,WeiT,XueF,LiGG,YangQ,etal.2016Regulationof417
Multi-drugResistanceinhepatocellularcarcinomacellsisTRPC6/CalciumDependent.Scientific418
Reports623269.419
YuY,WaliaV&ElbleRC2013LossofCLCA4PromotesEpithelial-to-MesenchymalTransitionin420
BreastCancerCells.PLoSONE8.421
ZhangJT,JiangXH,XieC,ChengH,DongJD,WangY,FokKL,ZhangXH,SunTT,TsangLL,etal.2013a422
DownregulationofCFTRpromotesepithelial-to-mesenchymaltransitionandisassociatedwithpoor423
prognosisofbreastcancer.BiochimicaEtBiophysicaActa-MolecularCellResearch18332961-2969.424
ZhangPS,YangXW,YinQ,YiJL,ShenWZ,ZhaoL,ZhuZ&LiuJW2016InhibitionofSK4Potassium425
ChannelsSuppressesCellProliferation,MigrationandtheEpithelial-MesenchymalTransitionin426
Triple-NegativeBreastCancerCells.PLoSONE11e0154471.427
ZhangSM,WangX,IqbalS,WangYR,OsunkoyaAO,ChenZJ,ChenZ,ShinDM,YuanHW,WangYQA,428
etal.2013bEpidermalGrowthFactorPromotesProteinDegradationofEpithelialProteinLostin429
Neoplasm(EPLIN),aPutativeMetastasisSuppressor,duringEpithelial-mesenchymalTransition.430
JournalofBiologicalChemistry2881469-1479.431
ZhengXF,CarstensJL,KimJ,ScheibleM,KayeJ,SugimotoH,WuCC,LeBleuVS&KalluriR2015432
Epithelial-to-mesenchymaltransitionisdispensableformetastasisbutinduceschemoresistancein433
pancreaticcancer.Nature527525-530.434
19
ZuoLA,LiW&YouSJ2010Progesteronereversesthemesenchymalphenotypesofbasalphenotype435
breastcancercellsviaamembraneprogesteronereceptormediatedpathway.BreastCancer436
Research12R34.437
438
439
440
441
20
442