Folia Biologica (Praha) 62, 131-137 (2016)

Original Article

Down-regulation of AQP4 Inhibits Proliferation, Migration and Invasion of Human Breast Cancer Cells(breast cancer / AQP4 / siRNA / E-cadherin / ERK pathway)

Y.-B. LI1, S.-R. SUN1, X.-H. HAN2

1Renmin Hospital of Wuhan University, Wuhan, China2Inner Mongolia People’s Hospital, Hohhot, China

Abstract. Aquaporins (AQPs), proteinaceous water channels, have been proposed as mediators of tu-mour development and progression. However, the role of aquaporin 4 (AQP4), a member of the AQP family, in breast cancer has not been distinctly evalu-ated. The aim of the present study was to examine the effect of AQP4 down-regulation on proliferation, migration and invasion in human breast cancer. To determine this effect, siRNA interference was used to knock down its expression in T47D and MCF-7 cell lines. Down-regulation of AQP4 resulted in increased expression of E-cadherin along with an inhibitory ef-fect on the proliferation, migration and invasion in breast cancer cells. In addition, AQP4 regulation of cell proliferation could be related with the ERK/E-cadherin pathway. In conclusion, the present data have suggested that down-regulation of AQP4 inhib-its breast cancer cell proliferation, migration and in-vasion.

IntroductionBreast cancer (BC) is estimated to be the most com-

monly diagnosed cancer in women, affecting millions of women worldwide (Siegel et al., 2014). Like other can-cers, breast cancer is a chronic and a multiple-step pro-cess in which accumulation of genetic and epigenetic alterations is involved. Although various achievements have been obtained in breast cancer research, the de-tailed understanding of its mechanism remains to be elu-cidated. To achieve a more effective treatment of human breast cancer, understanding the mechanisms that drive breast cancer progression is essential.

Aquaporins (AQPs) are a family of small hydropho-bic, integral membrane proteins ranging from 26–34 kDa in size and have been discovered in many organisms in-

Received September 3, 2015. Accepted November 2, 2015.

Corresponding author: Sheng-Rong Sun, Renmin Hospital of Wu-han University, Wuhan 430060, China. E-mail: shukege@163.com

Abbreviations: AQP – aquaporin, BC – breast cancer, siCtrl – control cells, siRNA – small interference RNA.

cluding mammals (Agre et al., 1993). At present, 13 transcellular water movement proteinaceous aquaporin (AQP) isoforms have been identified in mammals (Agre et al., 2002). Aquaporin 4 (AQP4), a member of the AQP family, has a key role in maintaining water and ion homeostasis, which is thought to be associated with tu-mour development (Verkman et al., 2008). AQP4 is known to participate mainly in the central nervous sys-tem disorders, such as brain oedema after injury or some other brain diseases (Siegel et al., 2014). Recent studies of AQP4 are not restricted to the central nervous system, and many researchers have found a new role for AQP4 in other biological processes. A previous study indicates that AQP4 knockout inhibits proliferation, migration and neuronal differentiation of adult neural stem cells derived from the subventricular zone of adult mice (Kong et al., 2008). Siegel et al. (2014) reported that AQP4 deficiency impairs migration and invasion of hu-man glioma cells. Shi et al. (2012) screened the expres-sion profiles of AQP0–12 in breast cancer tissues, and these results show a significantly higher level of AQP4 expression in normal than in cancer tissues, which im-plies that AQP4 may play a role in breast carcinogene-sis. However, up to date, there have been little reported attempts to study the relationship between AQP4 and breast cancer.

E-cadherin belongs to the cadherin family of cell ad-hesion molecules, playing an important role in the main-tenance of epithelial structure. Besides acting as an epi-thelial marker, E-cadherin also plays a role in controlling tumour growth and metastasis (Jeanes et al., 2008; Onder et al., 2008). Loss of E-cadherin has been frequently correlated with poor prognosis in human cancer and me-tastasis in the mouse model (Birchmeier and Behrens, 1994; Thiery, 2002). To get insight into the function of AQP4 in the mechanism of breast cancer development, further investigations are required. There fore, in this study, using down-regulation of AQP4 through small in-terference RNA (siRNA) we tested the hypothesis weather AQP4 may have a direct role in human breast cancer cell proliferation, migration and invasion. Our results may provide a new insight into the mechanism of breast cancer development.

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Material and MethodsCell culture and reagentsHumanbreastcancercelllinesMDA-MB-231,T47D,

MCF-7 and ER-ZR-70 were cultured in Dulbecco’sModified Eagle’s Medium (DMEM, Thermo FisherScientific Inc.,Waltham,MA) supplementedwith 1%pen/strepand10%FBS(BiologicalIndustries,KibbutzBeit-Haemek,Israel)andincubatedinahumidifiedatmosphereat37°Cin5%CO2. When they reached ~70-80%confluency,cellsweretrypsinizedandresus-pended in an antibiotic-free medium.

Small interference RNA (siRNA) treatmentT47DandMCF-7cellswereseededinto6-wellplates

for 24 h before transfection in the completemedium.Transient transfection was performed with Lipofectamine 2000 according to themanufacturer’s protocol (Invit-rogen,Carlsbad,CA).Humanbreastcancercellsweretransfectedwith5nMcontrolsiRNAorhumanAQP4siRNAoligos(5’-GCTCAATAGCTTTAGCAATTG-3’,GenechemCorp.,Shanghai,China).The transfectedcells were cultured in complete medium and incubated at37°Cfor24and48h.

RNA isolation, reverse transcription and real-time PCR

Total RNA was extracted using the RNeasy Kit(Qiagen,Hilden,Germany)accordingtothemanufactu-rer’s protocol. Reverse transcription was performed with 1μgtotalRNAperreactionusingthePrimeScriptRT-PCRKit (TakaraBio,Tepco,Japan).TheamountofcDNAequivalent to 5 ng total RNA was included in each PCR reaction.ExpressionanalysisofAQP4 and the house-keeping gene GAPDH was performed in T47D andMCF-7cellsbyquantitativereal-timePCR(ABIPrism7900HTSequenceDetectionSystem;AppliedBiosys-tems,Weiterstadt,Germany).Geneexpressiondifferen-ces between sample groups were analysed using t-test.

Western blotting analysisCells were lysed in ice-cold 1× RIPA lysis buffer

(containing50mMTris-HCl, pH7.4, 150mMNaCl,1%NP-40,1mMEDTA,0.25%sodiumdeoxycholate),1 mM NaF, 10mM Na3VO4, 1 mM PMSF (phenyl-methylsulphonylfluoride),andprotease inhibitorcock-tail(10mg/mlleupeptin,10mg/mlaprotinin,and1mMpepstatin). After incubation at 4 °C for 30 min, thelysates were centrifuged at 15,000 gfor10minat4°C.Protein concentrations were determined using a BCA protein assay kit, according to the manufacturer’s in-structions(PierceBiotechnology,Rockford,IL).Equalamountsofcell lysates(30μgtotalprotein/lane)wereloaded and separated in 12% SDS-PAGE and trans-ferredtoa0.45μmNCmembrane(Bio-RadLaboratories,Hercules, CA). The membranes were blocked in 5%non-fatdrymilkinTBS,pH7.4,for1h,thenimmuno-blottedwithprimaryantibodyovernightat4°C,andthe

primaryantibodieswereasfollows:anti-AQP4(SantaCruzBiotechnologysc-20812,SantaCruz,CA,1 : 1,000),anti-E-cadherin (Santa Cruz Biotechnology sc-7870,1 : 1,000),anti-p-Akt1(Thr308)(SantaCruzBiotech-nology sc-135650, 1 : 500), anti-p-ERK1/2 (Thr 202)(Santa Cruz Biotechnology sc-101760, 1 : 500) andβ-actin (Santa CruzBiotechnology sc1616, 1 : 1,000).Blotswerewashedwith0.1%Tween20inTBSandin-cubated in the secondary antibody for 1 h at room tem-perature. Protein intensities were determined and ana-lysed using Odyssey® Imager(LI-COR,Lincoln,NE).Theexpressionwascalculatedastheratioofeachspe-cificbandtoβ-actin.

Cell proliferation assayTheconfluentculturedT47DandMCF-7cellswere

trypsinizedandsuspendedinDMEMmediumwith10%FBS.Thecellswereplatedin96-wellplatesatadensityof1×104cellsperwellwithfreshmediumand/orsiRNA.Aftertreatmentfor24,48or72h,thecellproliferationwas determinedby theCellCountingKit-8 (Dojindo,Kyushu,Japan).Themediumofeachwellwasremovedandamixtureof10μlCCK-8and90μlof10%FBSRPMI-1640wasadded.Theplateswereincubatedforafurther3h,andtheabsorbanceat450nmwasmeasuredbyaBioTekElx808microplatereader(Winooski,VT).Five wells were read for all of the groups at each time point.Therelativecellsurvival(%)wasdeterminedbythefollowingformula:(ODsiRNA/ODscramble)×100%.

Wound healing assayHumanbreastcancercellswerestarved in1%FBS

culturemediumfor24handawoundwasmadewithaneven trace using a standard 200-μl pipette tip. Cellswereincubatedin5%CO2at37°Cfor24h,andimagesof the wound were captured at intervals. Wound healing wasquantifiedbymeasuringthemigratorydistanceofcells under a light microscope.

Transwell invasion assayCell invasion was measured by assessing the invasion

of cells through Matrigel-coated transwell inserts (Co-star,Cambridge,MA).Briefly,T47DandMCF-7cells(2×104)inculturemediumwith5%FBSwereloadedin theupper chamberof amodifiedBoydenchamber.The lower chamber contained culture medium including 20%FBSaschemoattractant.Cellswereincubatedfor24handthecellsremainingonthebottomsurfacewerefixed in 95% ethanol for 15 min, and stained withhaematoxylinandeosin.Observationswereperformedusingaphase-contrastmicroscopeinfivepredeterminedfields.

Statistical analysisStatistical analysis was carried out using Prizm 6

fromGraphPadSoftware (SanDiego,CA). Statisticalsignificanceforcomparisonbetweengroupswasdeter-mined by using Student’s t-test. All samples were tested intriplicate,andthedataareexpressedasmeans±SD.

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Results

Characterization of AQP4 expression in human breast cancer cell lines

WefirstexaminedtheexpressionofAQP4andE-cad-herininbreastcancercelllines(MDA-MB-231,T47D,MCF-7andER-ZR-70)byWesternblotting.AQP4wasexpressedinallfourhumanbreastcancercelllines,andtheexpressionofAQP4inT47Dwasthehighest.Thedifferences in E-cadherin expressionwere not signifi-cant(Fig.1A).WethereforechoseT47DandMCF-7cell lines for further investigation.

Down-regulation of AQP4 in human breast cancer cells by siRNA

First,wesoughttodown-regulatethemRNAexpres-sionofAQP4inT47DandMCF-7cellsusingsiRNAtechniques.AQP4 siRNA sequences were transfectedintoT47DandMCF-7cells,whichweredesignatedassiAQP4/T47Dand siAQP4/MCF-7 cells.A scrambledsiRNAsequencewasalsotransfectedtotheT47Dcells

and designated as scr/T47D cells. Control cells weredesignated as siCtrl cells. The transfected cells were screenedforAQP4expressionbyRT-PCRandWesternblottinganalyses.TheexpressionofAQP4mRNAandprotein was significantly reduced after silencing withsiRNAs(Fig.1B-E)

AQP4 modulates breast cancer cell proliferationTheresultsofthecellproliferationtestedbyCCK-8

showedthatAQP4-siRNA-transfectedT47DandMCF-7cellsexhibiteddecreasedcellgrowthability(P<0.05)comparedwiththecontrolgroup(Fig.2A-B,P<0.05).TheinhibitionrateofAQP4knockdownontheprolif-erationwas~25%.TheCCK-8experimentconfirmedthatT47DandMCF-7cellsweresensitivetodown-reg-ulationofAQP4,whichdisplayedan inhibitoryeffectontheproliferationofhumanT47Dcells(Fig.3).

AQP4 reduces breast cancer cell migration and invasion

Migration is a fundamental property of cells that oc-curs during many physiological and pathological pro-

RoleofAQP4inHumanBreastCancer

Fig. 1.Down-regulationofAQP4inhumanbreastcancercellsbysiRNA.(A)AQP4expressedinhumanbreastcancercell lines. (B-E)RT-PCRandWesternblotanalysisofAQP4expression insiCtrl, scr/T47DandsiAQP4/T47Dcells.β-actinwasusedasaloadingcontrol.

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cesses including organogenesis in the embryo, repair of damaged tissue after injury and the spread of cancer (Papadopoulosetal.,2008).Weexaminedthepotential

roleofAQP4incellmigrationaccordingtothehypo-thesis of water channel role in volume regulation associ-atedwithmigration.AsshowninFig.2,wefoundthatthe time required forwound closure ofAQP4-knock-downbreast cancerswas significantly longer than thetime required for the corresponding control cells. Our results obviously demonstrated that knockdown of AQP4greatly impaired themigratoryabilityofbreastcancercells(Fig.3).

Reduction of the cell migratory ability usually leads toreductionoftheirinvasiveability(Siegeletal.,2014).TodeterminewhetherAQP4,whichweshowedtoregu-late migration of breast cancer cells, also affects cell in-vasion, we assessed the invasiveness of the cells with AQP4 down-regulation in transwell assays.The inva-sionassayshowedsignificantdifferencesintheinvasiveabilitybetween the siAQP4cellsandsiCtrl cells (P<0.01;Fig.4).Quantitativeanalysisofthecellnumbersrevealed that siCtrl cells had a 2.3-foldhigher rate ofinvasionthansiAQP/T47Dcells,while theMCFcellsalso displayed 2.0-fold increase (Fig. 5).Collectively,theseresultsindicatethatAQP4playsanimportantrolein the migration and invasion of breast cancer cells.

Down-regulation of AQP4 enhances E-cadherin expression

E-cadherin is an important cell growth inhibitor (Stockinger et al., 2001).Given that ourdata indicateAQP4 regulationofcellgrowth inE-cadherin-expres-

Fig. 2. ScratchassayinT47DcellsandMCF-7cells.(A-B)siCtrlandsiAQP4cellsweregrowntoconfluenceina35-mmtissuedish,andthemonolayerwaswoundedwitha200-μlpipettetip.Imageswerecapturedat0,12hand24h.

Fig. 3. SilencingofAQP4significantlyinhibitsprolifera-tion in human breast cancer cell lines. (A-B)SilencingofAQP4 significantly inhibited proliferation ofT47D cellsandMCF-7cellsat12h,24h,48hcomparedtothecon-trol,asdeterminedbyCCK-8test(P<0.05).

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singcells,weattempted tounderstandwhetherAQP4regulatestheexpressionofE-cadherin.AsshowninFig.5A,knockdownofAQP4consistentlyincreasedE-cad-herinexpressionatboththetranscriptandproteinlevels;theseresultsindicatethattheexpressionofE-cadherinmayberegulatedbyAQP4inhumanbreastcancercells.IthasbeenreportedthattheexpressionofE-cadherin

canberegulatedthroughthePI3K/AKTorMAPK/ERKpathway. To investigate whether these pathways are af-fectedbyAQP4modulation,weknockeddownAQP4and determined the phosphorylation status of AKT and ERK1/2 inT47DandMCF-7 cells.As shown inFig.5B,wedidnotseeanyeffectofAQP4down-regulationon AKT phosphorylation. However, down-regulation of AQP4 resulted in increased phosphorylation of ERK1/2, indicating thatAQP4 is able to inhibit the ERKpathway in human breast cancer cells. These data sug-gestthatAQP4insufficiencymaypromotehumanbreastcancer cell growth through activation of the ERK path-way.

DiscussionAQPs have been shown to have pleiotropic biological

functions.AQP1andAQP4areinvolvedininvasionoflungcancercells (Xieetal.,2012).AQP2,3,4,5arerelatedwith the apoptosis process (Choi et al., 2009;Flamenco et al., 2009; Tang et al., 2011; Xie et al.,2013).AQP1couldpromotecellmigrationin4T1breastcancercells(Ridleyetal.,2003),andAQP5isamarker

protein for proliferation and migration of human breast cancer cellsMCF-7 (Xie et al., 2012).Recent studiesindicatedacorrelationofAQP4withgliomaprolifera-tion(Warthetal.,2004).AQP4isinvolvedinthecon-trol of glioblastoma cell migration and invasion (Amos etal.,2007).AQP4isdifferentiallyexpressedinbreastcancertissues(Shietal.,2012).However,littleisknownaboutthesignificanceofAQP4inbreastcancer.Thus,the aim of our study was to study the relationship be-tweenAQP4andbreastcancerdevelopment.Inthepresentstudy,wedecreasedtheexpressionlev-

elofAQP4inT47DandMCF-7cells.Theefficiencyandspecificityof thisdecreasewasconfirmedbyRT-PCRanalysis. Our results demonstrated that down-regulation ofAQP4inT47DandMCF-7cellscouldsignificantlyinhibitcellproliferation.ItseemsthatAQP4probablyfunctions as a positive regulator in breast cancer devel-opment.ReductionofAQP4canimpairtheT47DandMCF-7cellmigrationandinvasion.Cellmigrationisamultistep process involving numerous soluble growth factors, cytokines, proteases and extracellular matrixproteins(Ridleyetal.,2003).WefoundthatAQP4re-ductionslowsdownT47DandMCF-7cellmigrationincellchemotaxisandwoundhealingassays,suggestingthatAQP4isinvolvedinT47DandMCF-7cellmigra-tion.Similar tomigration, invasion through theextra-cellularmatrixisanimportantstepintumourinvasion(Amosetal.,2007).Itisalsostronglyassociatedwiththe infiltration and invasion of tumours (Maki et al.,2000).OurMatrigel invasionassay shows that reduc-

Fig. 4. Down-regulationofAQP4enhancesE-cadherinexpression. (A)Down-regulationofAQP4enhancedtheexpres-sion of E-cadherin in human breast cancer cells. (B)Down-regulationofAQP4resultedinincreasedphosphorylationofERK1/2.

RoleofAQP4inHumanBreastCancer

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tionofAQP4 led to adecreaseof invasiveness in theT47DandMCF-7cells.

Loss of E-cadherin function is a common event in cancer(Nolletetal.,1999).E-cadherinisknowntosup-press tumour cell growth and migration in various malig-nancies(YanagisawaandAnastasiadis,2006;Sotoetal.,2008).Wehavedemonstratedinthisstudythatknock-down of AQP4 can enhance E-cadherin expression.ERK kinases can activate several transcription factors andfurtherregulateexpressionofspecificgenes(Zhangetal.,2008;Hsuetal.,2011).Inourstudy,wefoundthatAQP4deficiencyup-regulatesE-cadherinexpressionbyactivationoftheERKpathway,asreflectedbythefind-ingthatAQP4knockdownenhancedphosphorylationofERK1/2.TheseresultssuggestthatAQP4maybeapo-tential therapeutic target of human breast cancer.

Understanding the molecular mechanisms of human breast cancer is critical for the development of novel

therapeutic strategies or treatments. In the present study, wediscovered the function ofAQP4 in humanbreastcancer cells. Hopefully, improved chemotherapy target-ingAQP4maybeavailabletopatientswithbreastcan-cerinthenextfewyears.

ConclusionIn this study, we provide evidence for a new role of

AQP4inbreastcancer.Ourresultsemphasizethatde-creasedAQP4expressioninhibitsproliferation,migra-tion and invasion of human breast cancer cells. Furt-hermore, knockdown of AQP4 expression enhancedE-cadherin expression and, at least in part,AQP4-re-gulated cell proliferation in breast cancer cells may be related with the ERK/E-cadherin pathway. In conclu-sion,thepresenteddatasuggestthatAQP4mayplayanimportant role in breast cancer.

Fig. 5. ReductionofAQP4bysiRNAimpairs the invasiveability in vitro. siCtrlandsiAQP4cells invading throughMatrigel-coated8-μmporesizetranswellinsertswereevaluatedasdescribedinmanufacturer’sinstructions.Thenumberofinvadingcellswasquantifiedbycountingthestainedcellsinrandomfieldsofthemembrane.

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Acknowledgement

Conflictsofinterest–none.

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RoleofAQP4inHumanBreastCancer