TTYH1 and TTYH2 Serve As LRRC8A-Independent Volume-Regulated Anion Channels in Cancer Cells

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Article TTYH1 and TTYH2 Serve as LRRC8A-Independent Volume-Regulated Anion Channels in Cancer Cells

1, 2,3, 1 4 1,2 Yeonju Bae †, Ajung Kim † , Chang-Hoon Cho , Donggyu Kim , Hyun-Gug Jung , Seong-Seop Kim 1, Jiyun Yoo 5, Jae-Yong Park 1,* and Eun Mi Hwang 2,3,* 1 College School of Biosystem and Biomedical Science of Health Science, Korea University, Seoul 02841, Korea; [email protected] (Y.B.); [email protected] (C.-H.C.); [email protected] (H.-G.J.); [email protected] (S.-S.K.) 2 Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea; [email protected] 3 KHU-KIST Department of Converging Science and Technology, Graduate School, Kyung Hee University, Seoul 02447, Korea 4 Namhae Garlic Research Institute, Gyeongnam 52430, Korea; [email protected] 5 Division of Applied Life Science (BK21 Plus), Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea; [email protected] * Correspondence: [email protected] (J.-Y.P.); [email protected] (E.M.H.) These authors contributed equally to this work. † Received: 21 May 2019; Accepted: 7 June 2019; Published: 9 June 2019

Abstract: Volume-regulated anion channels (VRACs) are involved in cellular functions such as regulation of cell volume, proliferation, migration, and cell death. Although leucine-rich repeat–containing 8A (LRRC8A) has been characterized as a molecular component of VRACs, here we show that Drosophila melanogaster tweety homologue 1 and 2 (TTYH1 and TTYH2) are critical for VRAC currents in cancer cells. LRRC8A-independent VRAC currents were present in the gastric cancer cell line SNU-601, but almost completely absent in its cisplatin-resistant derivative SNU-601-R10 (R10). The VRAC current in R10 was partially restored by treatment with trichostatin A (TSA), a histone deacetylase inhibitor. Based on microarray expression proﬁling of these cells, we selected two chloride channels, TTYH1 and TTYH2, as VRAC candidates. VRAC currents were completely absent from TTYH1- and TTYH2-deﬁcient SNU-601 cells, and were clearly restored by expression of TTYH1 or TTYH2. In addition, we examined the expression of TTYH1 or TTYH2 in several cancer cell lines and found that VRAC currents of these cells were abolished by gene silencing of TTYH1 or TTYH2. Taken together, our data clearly show that TTYH1 and TTYH2 can act as LRRC8A-independent VRACs, suggesting novel therapeutic approaches for VRACs in cancer cells.

Keywords: VRAC; TTYH1; TTYH2; LRRC8A; cancer cells

1. Introduction Volume-regulated anion channels (VRACs) are functionally expressed in almost all cell types, including cancer cells, and are intimately involved in regulation of cell volume, proliferation, migration, and death [1]. These channels open in response to osmotic swelling of the cell, and serve to restore the cell volume to its original state by releasing chloride ions and various organic osmolytes [2]. Attempts to identify the molecular components of VRACs have been ongoing for decades. In 2014, two research groups independently reported that leucine-rich repeat–containing 8A (LRRC8A) is a core component of VRAC [3,4]. Although LRRC8A has been reported to function as a VRAC in many diﬀerent cell types from a wide range of tissues, several studies suggested that it may not account for all VRAC functions. For

Cells 2019, 8, 562; doi:10.3390/cells8060562 www.mdpi.com/journal/cells Cells 2019, 8, 562 2 of 14 example, when LRRC8A is knocked down in HeLa cells, induced VRAC currents are diminished but still clearly present; moreover, knockdown does not affect cell death, and residual VRAC currents are still observed in LRRC8A-knockout HCT116 cells [5]. Another group showed that, in VRAC-deficient KCP-4 cells, VRAC currents are not restored by overexpression of LRRC8A alone or LRRC8A in combination with other LRRC8 isoforms [6]. In human retinal pigment epithelium (RPE) cells, bestrophin 1 (BEST1) is crucial for VRAC currents and volume regulation, and stable knockdown of LRRC8A has no effect [7]. Collectively, these studies suggested that channels that do not contain LRRC8A could also act as VRACs. The Drosophila melanogaster tweety (tty) gene, originally isolated as a transcription unit adjacent to flightless-1, encodes a large conductance chloride channel that belongs to a highly conserved and evolutionarily ancient family [8,9]. In 2004, human homologues of tweety (TTYH1–3) were identified and characterized: TTYH1 is a swelling-activated chloride channel, and TTYH2 and TTYH3 act as calcium-dependent maxi-chlorine channels when overexpressed in Chinese hamster ovary (CHO) cells [10]. To date, however, no study has examined VRAC activity in cells endogenously expressing TTYH1. In contrast to cisplatin-sensitive cancer cells, cisplatin-resistant cancer cells do not have VRAC currents in hypotonic solution [11,12]. In addition, when cisplatin-resistant cells are treated with trichostatin A (TSA), a histone deacetylase inhibitor, the VRAC currents are partially restored. Based on these observations, we predicted that the expression of genes responsible for VRAC activity should be altered in these cancer cells. Hence, to identify the gene(s) responsible for VRAC, we screened VRAC currents in several cancer cell lines and their cisplatin-resistant derivatives. Specifically, we examined the gastric cancer cell line SNU-601 and its cisplatin-resistant derivative SNU-601/Cis10 (R10). VRAC activity was absent in R10, but was restored by TSA treatment. Based on the gene expression profiles of these three types of cells (VRAC-active, VRAC-deficient, and VRAC-restored), we selected two candidate VRAC genes, TTYH1 and TTYH2, and confirmed that the channels they encode can act as VRACs in several types of cancer cells.

2. Materials and Methods

2.1. Cell Culture SNU-601, SNU-601/Cis10 (R10), and LoVo cells were grown in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA). HEK293T, HepG2, and MCF-7 cells were cultured in DMEM (Thermo Fisher Scientific). Culture medium was supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin–streptomycin, and cells were grown at 37 ◦C in a 5% CO2 incubator. The TTYH1 and TTYH2 double-knockout derivative of SNU-601 (dKO) was generated by Korea Bio (Seoul, Korea) using the CRISPR/Cas9 system (Santa Cruz, CA, USA). Knockouts of genes were confirmed by PCR and sequencing.

2.2. Microarray and Analysis Microarray analyses were performed using a commercial microarray service (Ebiogen, Seoul, Korea). Total RNA was isolated from SNU-601, R10, and R10_TSA cells using the RNA Puriﬁcation Kit (GeneAll, Seoul, Korea) and subjected to cDNA microarray analyses, performed by eBiogene (Seoul, Korea). Data normalization was performed using the GeneSpringGX7.3 software (Agilent Technology, Santa Clara, CA, USA). Brieﬂy, averages of normalized ratios were calculated by dividing normalized test channel intensities by normalized control channel intensities. Assessment of functional annotations and Gene Ontology (GO) was performed using GeneCards (https://www.genecards.org) web-based analysis. Cells 2019, 8, 562 3 of 14

2.3. Immunocytochemistry SNU-601 and dKO cells were grown on poly-D-lysine-coated coverslips. After washes in PBS, cells were ﬁxed in 4% paraformaldehyde in PBS for 15 min at room temperature, and then rinsed three times with PBS. Cells were permeabilized with 0.2% Triton X-100 in PBS for 7 min, and then incubated in blocking buﬀer (5% normal donkey serum, 3% BSA and 0.1% Triton X-100 in PBS) for 1 h. The cells were incubated with anti-TTYH2 antibody (Invitrogen, Carlsbad, CA, USA, PA5-34395) at 4 ◦C overnight. The next day, the cells were washed three times in PBS and incubated with suitable Alexa Fluor-tagged secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). After incubation with secondary antibodies, the cells were treated with phalloidin at room temperature for 30 min to label the plasma membrane. The coverslips were mounted on slides, and the cells were imaged by confocal microscopy (A1 confocal microscope; Nikon, Tokyo, Japan).

2.4. shRNA Construction and Validation Short hairpin shRNA (shRNA) vectors for human LRRC8A, TTYH1, and TTYH2 were constructed using pSicoR (Addgene, #11579) with GFP replaced by mCherry. shRNA sequences were as follows: LRRC8A: 50-GGTACAACCACATCGCCTA-30 [3]; TTYH1: 50-GCATCGGTTTCTATGGCAACA-30; TTYH2: 50-GGATTATCTGGACGCTCTTGC-30 [13]. A pSicoR construct containing a scrambled shRNA was used as a control. To validate shRNAs, SNU-601, HEK293T, HepG2, and LoVo cells were cultured and transfected with each shRNA in the presence of Lipofectamine 2000 (Invitrogen) in serum-free culture medium for 6 h, and then incubated in normal culture medium for 72 h. The eﬃciency of gene silencing was assessed by RT-PCR and western blot.

2.5. Western Blot For western blotting, HEK293T, SNU-601, and TTYH1/2 double-knockout cells were lysed with lysis buﬀer (50 mM HEPES, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF, and 0.1% SDS containing protease inhibitor cocktail, pH 7.4). Total protein (15 µg/lane) was subjected to SDS-PAGE (8–12% gels) and transferred to PVDF membranes. The membranes were blocked using 5% non-fat milk, and then blotted with the appropriate antibodies: Anti-LRRC8A (Cell Signaling Technology, Inc., Danvers, MA, USA, #24979), anti-TTYH1 (CUSABIO technology LLC, Houston, TX, USA, CSB-PA867139LA01HU), and anti-actin (Sigma, St. Louis, MO, USA, A5441). The membranes were then washed and incubated with HRP-conjugated goat anti-mouse (Jackson ImmunoResearch, #115-035-166), goat anti-rabbit (Jackson ImmunoResearch, #111-035-144), or rabbit anti-goat IgG (Jackson ImmunoResearch Lab, #305-035-045), followed by washing and detection of immunoreactivity using ECL (Thermo, #34095).

2.6. RT-PCR and Real-Time PCR Total RNA was isolated from cells using the RNA Puriﬁcation Kit from GeneAll. cDNAs were synthesized from 1 µg total RNA, and reverse transcription was performed using the SensiFAST™ cDNA Synthesis Kit (BIOLINE, London, UK). RT-PCR primer sequences were as follows: LRRC8A: Forward, 50-CTTCTCCTGAGTTCCTGGTC-30; reverse, 50-AAGGATGGCTCTGCTATCTG-30; TTYH1: Forward, 50-CTGGTGATCGTGATGACAGT-30; reverse, 50-TGCACCATAGTCCTTGTGCA-30; TTYH2: Forward, 50-GTGGACTACATCGCTCCCTG-30; reverse, 50-TGAACTTCAGGGTCTGCAGG-30; and GAPDH: Forward, 50-GTCTTCACCACCATGGAGAA-30; reverse, 50-GCATGGACTGTGGTCATGAG-30. GAPDH was used as a loading control. The LRRC8A, TTYH1 and TTYH2 fragments were ampliﬁed under the following cycle conditions: Denaturation at 94 ◦C for 30 s, annealing at 57 ◦C for 30 s, and extension at 72 ◦C for 30 s. This cycle was repeated a total of 32 times. GAPDH fragments were also amplified under the same conditions except that 24 cycles were run. Real-time PCR was performed using the SensiFAST™ Probe Hi-ROX kit (Invitrogen). Primer sets for LRRB8A (Hs.PT.58.346894), LRRC8B (Hs.PT.58.12310), LRRC8C (Hs.PT.58.1804454), LRRC8D (Hs.PT.58.15346618), LRRC8E Cells 2019, 8, 562 4 of 14

(Hs.PT.58.48538734), TTYH1 (Hs.PT.58.40149255), TTYH2 (Hs.PT.58.4787185), TTYH3 (Hs.PT.58.21128423), CFTR (Hs.PT.58.3365414), and GAPDH (Hs.PT.39a.22214836) were generated by IDT (PrimeTime qPCR -∆∆C assays). All experiments were repeated at least three times. The 2 T method was used to calculate fold changes in gene expression [14].

2.7. Electrophysiology The standard pipette solution contained the following (in mM): 60 Trizma-HCl, 70 Trizma-base, 70 aspartic acid, 15 HEPES, 0.4 CaCl2, 1 MgCl2, 1 EGTA, 1 ATP, and 0.5 GTP; the pH was adjusted to 7.2 with CsOH. The bath contained the following (in mM): 70 Trizma-HCl, 1.5 CaCl2, 10 HEPES, 10 D-glucose, and 100 sucrose (290 mOsm/kg); the pH was adjusted to 7.4 with CsOH. To block K+ currents, 5 mM TEA and 5 mM BaCl2 were added to the bath solution. Hypotonic solution had the same ionic composition as the bath solution but lacked sucrose (220 mOsm/kg; the pH was adjusted to 7.4 with CsOH). Patch pipettes were made from borosilicate glass capillaries (Warner Instruments, Hamden, CT, USA), and had a resistance of 5–6 MΩ. Whole-cell currents were recorded using a patch-clamp amplifier (Axopatch 700B; Molecular Devices, Silicon Valley, CA, USA). Current–voltage relationships were measured by applying ramp pulses (from 100 mV to +100 mV, 1 s duration) from a − holding potential of 60 mV. A Digidata 1550A interface was used to convert digital–analogue signals. − Data were sampled at 5 kHz and filtered at 1 kHz. Currents were analyzed with the Clampfit software (Molecular Devices). All experiments were conducted at room temperature.

2.8. Statistical Analysis All statistical analyses were performed using GraphPad Prism version 8.00 (GraphPad Software, La Jolla, CA, USA) for Windows. Numerical data are presented as means standard error of the mean ± (SEM). Statistical differences were evaluated by ANOVA and the paired or unpaired Student’s t-test, as appropriate. Differences were considered significant at P < 0.05.

3. Results

3.1. VRAC Currents are Shown in SNU-601 Cells but not in Cisplatin-Resistant R10 cells To observe VRAC activity, we used whole-cell patch-clamp recording in the gastric cancer cell line SNU-601 and its cisplatin-resistant derivative SNU-601/Cis10 (R10). R10 cells were generated by chronic exposure to 10 mg/mL cisplatin, a platinum-containing anti-cancer drug [15]. In hypotonic solution, VRAC-like currents were gradually induced in SNU-601 cells that were similar to those observed in other cancer cells [11], but no current was detected in R10 cells. In addition, the current–voltage (I–V) relationship indicated that the ICl currents in SNU-601 cells were dramatically increased in response to hypotonic solution. However, the I–V relationship of ICl currents remained almost unchanged in R10 cells (Figure1b,c). To determine whether the hypotonicity-induced I Cl currents in SNU-601 cells were VRAC currents, we treated cells with DCPIB, a selective blocker of VRAC [16,17]. The elevated ICl currents in hypotonic solution were inhibited in 30 µM DCPIB (Figure1d,e). These ﬁndings suggest that SNU-601 gastric cancer cells have volume-regulated ICl currents, whereas cisplatin-resistant R10 cells do not. Cells 2019, 8, x FOR PEER REVIEW 5 of 14 Cells 2019, 8, 562 5 of 14

Figure 1. Volume-activated chloride currents in SNU-601 cells. (a) Representative traces showing timeFigure courses 1. Volume-activated of the volume-activated chloride currents chloride in currentSNU-601 in cells. SNU-601 (a) Representative and R10 cells traces elicited showing by voltage time rampcourses from of the100 volume-activated to +100 mV. (b chloride) Representative current in traces SNU-601 showing and R10 the cells current–voltage elicited by voltage relationship ramp − forfrom volume-activated −100 to +100 mV. chloride(b) Representative currents in traces SNU-601 showing and the R10 cu cellsrrent–voltage before and relationship during perfusion for volume- with hypotonicactivated chloride solution, currents respectively. in SNU-601 (c) Summary and R10 bar cell graphs before showing and during the ratio perfusion of current with amplitudes hypotonic ofsolution, SNU-601 respectively. (n = 7) and (c) R10Summary cells(n bar= graph7) before showing and duringthe ratio perfusion of current with amplitudes a hypotonic of SNU-601 solution. (n (=d 7)) Representative and R10 cells (n traces = 7) ofbefore volume-regulated and during perfusion anion channel with a (VRAC) hypotonic currents solution. of SNU-601 (d) Representative cells before andtraces during of volume-regulated perfusion with a hypotonicanion channel solution, (VRAC) and during currents DCPIB of SNU-601 application cells in a before hypotonic and solution. during (perfusione) Summary with bar a graph hypotonic showing solution, the ratio and of currentduring amplitudesDCPIB application of DCPIB-sensitive in a hypotonic currents solution. before and (e) afterSummary DCPIB bar application graph showing (n = 7). the Data ratio are of presentedcurrent am asplitudes means ofSEM DCPIB-sensitive (*** P < 0.001). currents before and ± after DCPIB application (n = 7). Data are presented as means ± SEM (*** P < 0.001). 3.2. SNU-601 Cells Have LRRC8A-Independent VRAC Currents 3.2. SNU-601Previous Cells studies Have showed LRRC8A-Independent that LRRC8A (SWELL1) VRAC Currents is a key component of the VRAC [3,4]. Therefore, we firstPrevious investigated studies whether showed the that hypotonicity-induced LRRC8A (SWELL I1)Cl currentsis a key incomponent SNU-601 cells of the were VRAC dependent [3,4]. onTherefore, LRRC8A. we To first this investigated end, we constructed whether the a shRNA hypotonicity-induced against LRRC8A ICl and currents confirmed in SNU-601 that it ecellsfficiently were silenceddependent LRRC8A on LRRC8A. expression To this in end, SNU-601 we constructe cells (Supplementaryd a shRNA against Materials LRRC8A Figure and S1).confirmed In SNU-601 that it cellsefficiently transfected silenced with LRRC8A LRRC8A expression shRNA, hypotonicity-inducedin SNU-601 cells (Supplementary VRAC currents Materials were comparable Figure 1). toIn thoseSNU-601 in SNU-601 cells transfected cells transfected with LRRC8A with control shRNA, scrambled hypotonicity-induced shRNA (Figure2a,b). VRAC Because currents thisresult were wascomparable unexpected, to those weexamined in SNU-601 VRAC cells currents transfecte in HEK293Td with control cells, inscrambled which LRRC8A shRNA was(Figure originally 2a,b). identifiedBecause this as aresult VRAC was component unexpected, [3]. we In HEK293examined cells VRAC transfected currents with in HEK293T LRRC8A shRNA,cells, in VRACwhich currentsLRRC8A were was notoriginally induced identified in hypotonic as a solution, VRAC ascomponent previously [3]. reported In HEK293 (Figure cells2c,d). transfected with LRRC8ABecause shRNA, LRRC8A VRAC has currents four closely were related not induced homologues in hypotonic (LRRC8B–E) solution, and formsas previously heteromers reported [4,18], we(Figure examined 2c,d). the expression levels of the five LRRC8 family members in SNU-601 and R10 cells by quantitativeBecause RT-PCRLRRC8A (qRT-PCR) has four closely (Figure related2e). Relative homologues expression (LRRC8B–E) levels and of LRRC8A, forms heteromers LRRC8D, [4,18], and LRRC8Ewe examined were the unchanged expression between levels of SNU-601 the five andLRRC8 R10 family cells, but members LRRC8B in wasSNU-601 higher and in R10 R10. cells These by dataquantitative suggested RT-PCR that expression (qRT-PCR) of the(Figure other 2e). LRRC8 Relative family expression members levels was notof LRRC8A, correlated LRRC8D, with cisplatin and resistanceLRRC8E were in these unchanged cells. Taken between together, SNU-601 these and findings R10 cells, indicate but LRRC8B that hypotonicity-induced was higher in R10. VRACThese currentsdata suggested in SNU-601 that expression cells do not of depend the other on LRRC8 LRRC8A family expression. members was not correlated with cisplatin resistance in these cells. Taken together, these findings indicate that hypotonicity-induced VRAC currents in SNU-601 cells do not depend on LRRC8A expression.

CellsCells2019 2019, 8, ,8 562, x FOR PEER REVIEW 66 ofof 14 14

Figure 2. SNU-601 cells have a LRRC8A-independent VRAC activity. (a) Representative traces showing theFigure current–voltage 2. SNU-601 relationship cells have fora LRRC8A-ind VRACs in SNU-601ependent cells VRAC transfected activity. with (a scrambled) Representative or LRRC8A traces shRNAsshowing under the current–voltage isotonic or hypotonic relationship conditions. for VRAC (b) Summarys in SNU-601 bar graphcells transfected showing the with ratio scrambled of current or amplitudesLRRC8A shRNAs of hypotonic under/isotonic isotonic solutions or hypotonic in SNU-601 conditions. cells (b transfected) Summary with bar graph scrambled showing or LRRC8A the ratio shRNAsof current (n amplitudes= 6). (c) Representative of hypotonic/isotonic traces showing solutions the in current–voltageSNU-601 cells transfected relationship with for scrambled VRACs in or HEK293TLRRC8A cellsshRNAs transfected (n = 6). with (c) scrambled Representative or LRRC8A traces shRNAs showing under the isotoniccurrent–voltage or hypotonic relationship conditions. for (dVRACs) Summary in HEK293T bar graph cells showing transfec theted ratio with of current scrambled amplitudes or LRRC8A of hypotonic shRNAs/isotonic under solutions isotonic inor SNU-601hypotonic cells conditions. transfected with(d) Summary scrambled shRNAbar graph (n = 5)showing or LRRC8A the shRNAratio (nof =current11). (e) Real-timeamplitudes PCR of quantificationhypotonic/isotonic of fold solutions changes in in LRRC8 SNU-601 family cells mRNAs transfected in SNU-601 with scram and R10bled cells. shRNA The experiments(n = 5) or LRRC8A were repeatedshRNA three(n = 11). times. (e) DataReal-time are presented PCR quantification as means ofSEM fold (** changesP < 0.01, in *** LRRC8P < 0.001, family n.s, mRNAs not significant). in SNU- ± 601 and R10 cells. The experiments were repeated three times. Data are presented as means ± SEM (** 3.3. Whole-Genome Screening Identifies TTYH1 and TTYH2 as VRAC Candidates P < 0.01, *** P < 0.001, n.s, not significant). TSA is a histone deacetylase inhibitor and potent anti-cancer agent [19]. A previous study showed that3.3. TSA Whole-Genome partially restored Screening VRAC Identifies currents TTYH1 in KCP-4 and TTYH2 cells [11 as]. VRAC Therefore, Candidates we investigated whether TSA treatmentTSA couldis a histone restore VRACdeacetylase currents inhibitor in cisplatin-resistant and potent anti-cancer R10 cells. Asagent shown [19]. in A Figure previous1a, VRAC study currentsshowed inthat R10 TSA cells partially were not restored induced VRAC in hypertonic currents solution,in KCP-4 but cells were [11]. restored Therefore, in R10 we cells investigated treated withwhether TSA (FigureTSA treatment3a). To identifycould restore candidate VRAC genes currents encoding in cisplatin-resistant VRAC components R10 or cells. their As regulators shown in inFigure SNU-601 1a, VRAC cells, we currents used Whole in R10 Human cells were Genome not indu Microarraysced in hypertonic (44,000 genes)solution, to monitorbut were expression restored in levelsR10 cells in SNU-601, treated with R10, andTSA TSA-treated (Figure 3a). R10 To identi cells. Wefy candidate initially focused genes encoding on proteins VRAC predicted components to have at or leasttheir one regulators transmembrane in SNU-601 (TM) cells, domain we (6081used genes)Whole (Figure Human3b). Genome From this Microarrays list, we identified (44,000 chloridegenes) to channelsmonitor (95expression genes, Supplementary levels in SNU-601, Table S1), R10, and and then TSA-treated selected three R10 candidate cells. We genes initially (TTYH1, focused TTYH2, on proteins predicted to have at least one transmembrane (TM) domain (6081 genes) (Figure 3b). From

Cells 2019, 8, x FOR PEER REVIEW 7 of 14 this list, we identified chloride channels (95 genes, Supplementary Table S1), and then selected three candidate genes (TTYH1, TTYH2, and CFTR) that were expressed in R10 cells at 20% of the level in SNU-601 cells. Of those, only two (TTYH1 and TTYH2) were more than 2-fold upregulated in TSA- treated R10 cells relative to untreated R10 cells (Figure 3b). Initially, we generated scatter plots of log (read numbers) in SNU-601 cells vs. R10 cells (Figure 3c, left). This analysis confirmed that TTYH1, TTYH2, and CFTR genes were down-regulated in R10 cells. Next, we compared the scatter plots representing the log (read numbers) in R10 cells vs. TSA- treated R10 cells (Figure 3c, right), and found that the level of CFTR expression was still low in TSA- treated R10 cells. In addition, we confirmed the relative levels of TTYH1, TTYH2, and CFTR in three different cell groups using qRT-PCR (Figure 3e). The mRNA levels of TTYH1 and TTYH2 were Cellssignificantly2019, 8, 562 reduced in R10 cells, but recovered in TSA-treated R10 cells. In the case of CFTR,7 ofthe 14 mRNA level was reduced in R10 cells, but did not recover in TSA-treated R10 cells. Expression of TTYH3, another TTYH family member, was highest in R10 cells, indicating that TTYH3 may not and CFTR) that were expressed in R10 cells at 20% of the level in SNU-601 cells. Of those, only two function as a VRAC (Supplementary Materials Figure 2). Together, these results identified TTYH1 (TTYH1 and TTYH2) were more than 2-fold upregulated in TSA-treated R10 cells relative to untreated and TTYH2 as a VRAC candidate. R10 cells (Figure3b).

Figure 3. TTYH1 and TTYH2 are candidate VRACs in SNU-601 cells. (a) Representative traces showing the time course of volume-activated chloride currents elicited by alternating pulses by voltage ramp

from 100 to +100 mV, in R10 and TSA-treated R10 cells. (b) Schematic overview of our human − whole-genome array screen to identify candidate VRAC components. (c) Scatter plots of log2 (Raw) for every gene in SNU-601 vs. R10 and TSA-treated R10 vs. R10. (d) Dot plots of expression levels of TTYH1, TTYH2, and CFTR in SNU-601, R10, and TSA-treated R10 cells samples, calculated rom array data. (e) Real-time PCR quantification of fold changes in TTYH1, TTYH2, and CFTR mRNAs in SNU-601, R10, and TSA-treated R10 cells. Data are presented as means SEM (** P < 0.01). ± Initially, we generated scatter plots of log (read numbers) in SNU-601 cells vs. R10 cells (Figure3c, left). This analysis confirmed that TTYH1, TTYH2, and CFTR genes were down-regulated in R10 cells. Next, we compared the scatter plots representing the log (read numbers) in R10 cells vs. TSA-treated Cells 2019, 8, x FOR PEER REVIEW 8 of 14 Cells 2019, 8, 562 8 of 14 Figure 3. TTYH1 and TTYH2 are candidate VRACs in SNU-601 cells. (a) Representative traces showing the time course of volume-activated chloride currents elicited by alternating pulses by R10 cellsvoltage (Figure ramp3 c,from right), −100 and to +100 found mV, that in R10 the and level TSA-treated of CFTR expression R10 cells. ( wasb) Schematic still low overview in TSA-treated of our R10 cells.human In addition, whole-genome we confirmed array screen the relative to identify levels candidate of TTYH1, VRAC TTYH2, components. and CFTR (c) Scatter in three plots di offf erentlog2 cell groups(Raw) using forqRT-PCR every gene (Figure in SNU-6013e). The vs. mRNA R10 and levels TSA-treated of TTYH1 R10 and vs. TTYH2R10. (d) wereDot plots significantly of expression reduced in R10levels cells, ofbut TTYH1, recovered TTYH2, in and TSA-treated CFTR in SNU-601, R10 cells. R10, In theand caseTSA-treated of CFTR, R10 the cells mRNA samples, level calculated was reduced in R10rom cells, array but data. did not(e) Real-time recover in PCR TSA-treated quantification R10 cells.of fold Expression changes in of TTYH1, TTYH3, TTYH2, another and TTYH CFTR family member,mRNAs was in highest SNU-601, in R10R10, cells,and TSA-treated indicating R10 that ce TTYH3lls. Data may are presented not function as means as a VRAC ± SEM (Supplementary(** P < 0.01). Materials Figure S2). Together, these results identified TTYH1 and TTYH2 as a VRAC candidate. 3.4. TTYH1/TTYH2 dKO Cells Lack VRAC Currents 3.4. TTYH1/TTYH2 dKO Cells Lack VRAC Currents To further investigate whether TTYH1 and TTYH2 are responsible for hypotonicity-induced VRACTo currents, further investigatewe used TTYH1/TTYH2 whether TTYH1 double-k and TTYH2nockout are (dKO) responsible cells generated for hypotonicity-induced by CRISPR/Cas9 VRACgene editing. currents, Exon we 7 usedof TTYH1 TTYH1 and/TTYH2 exons 2 double-knockout and 3 of TTYH2 were (dKO) targeted, cells generated and the double by CRISPR knockout/Cas9 wasgene verified editing. by Exon western 7 of TTYH1blotting. and As exonsshown 2 in and Figu 3 ofre TTYH2 4a, expression were targeted, of TTYH1 and was the abolished double knockout in dKO wascells, verified whereas by LRRC8A western blotting.expression As was shown unchange in Figured. 4Ina, expressionimmunocytochemical of TTYH1 was staining, abolished TTYH2 in dKO was notcells, detected whereas in LRRC8A the dKO expressioncells (Figure was 4b). unchanged. To determine In immunocytochemicalwhether dKO cells had staining, VRAC TTYH2activity, was we measurednot detected hypotonicity-induced in the dKO cells (Figure VRAC4b). currents To determine in dKO whether cells transfected dKO cells with had VRACGFP control, activity, GFP- we taggedmeasured TTYH1, hypotonicity-induced and GFP-tagged VRAC TTYH2 currents (Figure in dKO4c–g). cells In transfectedthe GFP control–transfected with GFP control,GFP-tagged dKO cells, TTYH1,VRAC currents and GFP-tagged were not TTYH2induced (Figure in hypotonic4c–g). In solution the GFP (Figure control–transfected 4c). By contrast, dKO in cells, dKO VRAC cells transfectedcurrents were with not inducedGFP-tagged in hypotonic TTYH1 solutionor GFP-tagg (Figureed4 c).TTYH2, By contrast, VRAC in dKOcurrents cells transfectedwere efficiently with producedGFP-tagged under TTYH1 hypotonic or GFP-tagged conditions TTYH2, (Figure VRAC 4d,e). currents However, weree wefficiently observed produced no additive under hypotoniceffects in dKOconditions cells co-transfected (Figure4d,e). with However, both TTYH1 we observed and TTYH2 no additive (Figure e ff4f).ects Together, in dKO cellsthese co-transfected data indicatedwith that both TTYH1 and TTYH2 (Figurecan serv4f).e as Together, VRACs in these SNU-601 data indicated cells. that both TTYH1 and TTYH2 can serve as VRACs in SNU-601 cells.

Figure 4. VRAC activity is completely abolished in dKO cells, and is restored by expression of TTYH1 Figureor TTYH2. 4. VRAC (a) Western activity is blot completely data for TTYH1abolished and in LRRC8AdKO cells, (SWELL1) and is restored in SNU-601 by expression and TTYH1 of TTYH1 and TTYH2or TTYH2. double-knockout (a) Western blot (dKO) datacells. for TTYH1 (b) Representative and LRRC8A immunocytochemical (SWELL1) in SNU-601 images and TTYH1 of SNU-601 and TTYH2and dKO double-knockout cells stained with (dKO) anti-TTYH2 cells. (b antibody.) Representative (c) Representative immunocyto traceschemical of VRAC images currents of SNU-601 from anddKO dKO cells cells transfected stained with with GFP anti-TTYH2 controls inantibody. isotonic and(c) Representative hypotonic solutions. traces Notably,of VRAC VRAC currents currents from weredKO cells not induced transfected by hypotonicwith GFP controls stimulation in isotonic in these and cells. hypotonic (d) Representative solutions. Notably, traces of VRAC currents werefrom dKOnot induced cells transfected by hypotonic with stimulation TTYH1-GFP in inthese isotonic cells. and (d) Representative hypotonic solutions. traces (ofe) VRAC Representative currents fromtraces dKO of VRAC cells transfected currents from with dKO TTYH1-GFP cells transfected in isotonic with and TTYH2-GFPhypotonic solutions. in isotonic (e) andRepresentative hypotonic tracessolutions. of VRAC (f) Representative currents from traces dKO of VRACcells transfec currentsted from with dKO TTYH2-GFP cells co-transfected in isotonic with and TTYH1-GFP hypotonic solutions.and TTYH2-GFP (f) Representative in isotonic and traces hypotonic of VRAC solutions. currents ( gfrom) Summary dKO cells bar co-transfected graph showing with current TTYH1-GFP densities andin isotonic TTYH2-GFP and hypotonic in isotonic solutions, and hypotonic as in (c)–( solutions.f), at +100 (g mV.) Summary Data are bar presented graph asshowing means currentSEM ± (**densitiesP < 0.01 in andisotonic *** P and< 0.001). hypotonic solutions, as in (c)–(f), at +100 mV. Data are presented as means ± SEM (** P < 0.01 and *** P < 0.001). 3.5. TTYH1 and TTYH2 Have Independent VRAC Activity in Other Cancer Cell Lines To establish the VRAC activity of TTYH1 and TTYH2, we investigated other cancer cell lines that endogenously express these two channels. Among the cell lines we screened, HepG2 cells expressed

Cells 2019, 8, x FOR PEER REVIEW 9 of 14

Cells 20193.5., 8TTYH1, 562 and TTYH2 Have Independent VRAC Activity in Other Cancer Cell Lines 9 of 14 To establish the VRAC activity of TTYH1 and TTYH2, we investigated other cancer cell lines that endogenously express these two channels. Among the cell lines we screened, HepG2 cells TTYH1, but not TTYH2, whereas LoVo cells exhibited the opposite expression pattern (Figure5a), and expressed TTYH1, but not TTYH2, whereas LoVo cells exhibited the opposite expression pattern MCF-7 cells expressed neither mRNA. Next, we asked whether single expression of the TTYH isoform (Figure 5a), and MCF-7 cells expressed neither mRNA. Next, we asked whether single expression of wouldthe be TTYH suﬃ cientisoform for would hypotonicity-induced be sufficient for hypotonicity-induced VRAC activity in each VRAC cell activity type (Figure in each5 b–e).cell type VRAC currents(Figure were 5b–e). induced VRAC in currents hypotonic were solution induced in in both hypotonic HepG2 solution and LoVo in both cells, HepG2 and were and almost LoVo cells, inhibited by 30andµM were DCPIB almost (Figure inhibited5b–e). by 30 On μM the DCPIB other (Figure hand, 5b–e). MCF-7 On cells the ot hadher veryhand, small MCF-7 VRAC cells had currents very in hypotonicsmall VRAC solution, currents and thesein hypotonic were suppressed solution, and by these DCPIB were (Figure suppressed5f,g). by DCPIB (Figure 5f,g).

FigureFigure 5. VRAC 5. VRAC currents currents are are observed observed in in cancer cancer cellcell lines expressing expressing TTYH1 TTYH1 or TTYH2. or TTYH2. (a) Expression (a) Expression of TTYH1of TTYH1 and TTYH2 and TTYH2 mRNA mRNA in various in various cancer cellcancer lines cell was lines measured was measured by RT-PCR. by (bRT-PCR.) Representative (b) tracesRepresentative of VRAC currents traces from of VRAC HepG2 currents cells infrom isotonic HepG2 and cells hypotonic in isotonic solutions. and hypotonic (c) Summary solutions. bar (c graph) showingSummary current bar densitiesgraph showing of HepG2 current cells densities in isotonic of HepG and2 cells hypotonic in isotonic solutions, and hypotonic as in solutions, (b), at +100 as mV. ( d) Representative traces of VRAC currents from LoVo cells in isotonic and hypotonic solutions. (e) Summary bar graph showing current densities of LoVo cells in isotonic and hypotonic solutions, as in (d), at +100 mV. (f) Representative traces of VRAC currents from MCF-7 cells in isotonic and hypotonic solutions. (g) Summary bar graph showing current densities of MCF-7 cells in isotonic and hypotonic solutions, as in (f), at +100 mV. Data are presented as means SEM (* P < 0.05, ** P < 0.01, ± **** P < 0.0001). Cells 2019, 8, x FOR PEER REVIEW 10 of 14

in (b), at +100 mV. (d) Representative traces of VRAC currents from LoVo cells in isotonic and hypotonic solutions. (e) Summary bar graph showing current densities of LoVo cells in isotonic and hypotonic solutions, as in (d), at +100 mV. (f) Representative traces of VRAC currents from MCF-7 cells in isotonic and hypotonic solutions. (g) Summary bar graph showing current densities of MCF- 7 cells in isotonic and hypotonic solutions, as in (f), at +100 mV. Data are presented as means ± SEM

Cells 2019(* P, 8<, 0.05, 562 ** P < 0.01, **** P < 0.0001). 10 of 14

We next investigated the effect of TTYH1 or TTYH2 silencing on VRAC currents in HepG2 and LoVoWe cells. next The investigated level of TTYH1 the eff mRNAect of TTYH1 was significantly or TTYH2 silencingreduced by on TTYH1 VRAC currentsshRNA in in HepG2 HepG2 cells and LoVo(Figure cells. 6a).The The level VRAC of TTYH1currents mRNA induced was in significantlythe hypotonic reduced solution by were TTYH1 dramatically shRNA in decreased HepG2 cells in (FigureHepG26 cellsa). The transfected VRAC currents with TTYH1 induced shRNA in the relative hypotonic to those solution in cells were treated dramatically with scrambled decreased shRNA in HepG2(Figure cells6b,c). transfected Next, we examined with TTYH1 the effect shRNA of TTY relativeH2 shRNA to those in in LoVo cells cells. treated TTYH2 with scrambledshRNA effectively shRNA (Figuredecreased6b,c). the Next, expression we examined level of the TTYH2 e ffect relative of TTYH2 to that shRNA in cells in LoVo transfected cells. TTYH2 with scrambled shRNA eff ectivelyshRNA decreased(Figure 6d), the and expression suppressed level most of TTYH2VRAC currents relative to(Fig thature in 6e,f). cells Taken transfected together, with these scrambled results shRNAsuggest (Figurethat TTYH16d), and and suppressed TTYH2 function most VRACindependen currentstly as (Figure VRACs6e,f). in various Taken together, cancer cells. these results suggest that TTYH1 and TTYH2 function independently as VRACs in various cancer cells.

Figure 6. Lack of TTYH1 or TTYH2 alone in HepG2 or LoVo cells almost completely eliminates VRACFigure activity.6. Lack of ( aTTYH1)Eﬃciency or TTYH2 of gene alone silencing in HepG2 by or the Lo TTYH1Vo cells shRNA almost constructcompletely was eliminates monitored VRAC by RT-PCRactivity. in(a)HepG2 Efficiency cells. of gene (b) Representative silencing by the traces TTYH1 of VRAC shRNA currents construct from was HepG2 monitored cells transfectedby RT-PCR eitherin HepG2 with cells. scrambled (b) Representative or TTYH1 shRNA traces inof isotonicVRAC currents and hypotonic from HepG2 solutions. cells ( ctransfected) Summary either bar graph with showingscrambled current or TTYH1 densities shRNA of transfected in isotonic HepG2 and hypotonic cells in isotonic solutions. and (c hypotonic) Summary solutions, bar graph as inshowing (b), at +current100 mV. densities (d)Eﬃciency of transfected of gene silencingHepG2 cells by thein isotonic TTYH2 and shRNA hypotonic construct solutions, was monitored as in (b), at by +100 RT-PCR. mV. ((ed)) RepresentativeEfficiency of gene traces silencing of VRAC by currents the TTYH2 from shRNA LoVo cells construct transfected was monitored with scrambled by RT-PCR. or TTYH2 (e) shRNARepresentative in isotonic traces and of hypotonic VRAC currents solutions. from (f )Lo SummaryVo cells bartransfected graph showing with scrambled current densities or TTYH2 of transfectedshRNA in isotonic LoVo cells and in isotonichypotonic and solutions. hypotonic (f solutions,) Summary as bar in ( egraph), at + 100showing mV. Data current are presenteddensities asof means SEM (** P < 0.01, **** P < 0.0001). transfected± LoVo cells in isotonic and hypotonic solutions, as in (e), at +100 mV. Data are presented 4. Discussionas means ± SEM (** P < 0.01, **** P < 0.0001). VRACs play important roles in regulation of cell volume, proliferation, migration, and death [1]. In particular, VRACs are also involved in the responsiveness of cancers to Pt-based anti-cancer drugs such as cisplatin and carboplatin [20]. LRRC8A acts as a core component of VRAC in diverse cell types [3,4], but VRAC activity is still present in LRRC8A-knockout HCT116 and LRRC8A-knockdown HeLa cells [5]. These data raise the possibility that other channels can also act as VRACs. Cells 2019, 8, 562 11 of 14

In this study, we found that the VRAC currents of SNU-601 cells were largely unaffected by silencing of LRRC8A, even though this gene is efficiently expressed in SNU-601 cells (Figure2). Consistent with a previous study of RPE cells [7], our data confirmed that LRRC8A-independent VRAC currents can be detected in this cancer cell line. In addition, the VRAC currents were almost completely abolished in cisplatin-resistant R10 cells, but were partially restored upon TSA treatment (Figures1 and3). We used SNU-601, R10, and TSA-treated R10 cells to identify the LRRC8A-independent VRAC(s). Our strategy for finding new VRACs was based on the reasoning that, if a specific gene encoding a LRRC8A-independent VRAC exists, its expression should be maintained in SNU-601 cells, suppressed in R10 cells, and recovered in TSA-treated R10 cells. Normally, when we compare changes in gene expression, each gene is classified into one of three groups (increase, no change, or decrease). However, if changes in gene expression are considered under three different conditions, each gene must be assigned to one of nine groups; consequently, only a small number of candidates can be selected. Based on microarray expression profiles of SNU-601, R10, and TSA-treated R10 cells, we defined a short list of candidate genes whose expression levels were correlated with VRAC currents in these three cell types (Figure3). Our microarray data also clearly showed that not all LRRC8 isoforms’ expression patterns matched VRAC activities in three different cells (VRAC-active, VRAC-deficient, and VRAC-restored). Likewise, the levels of mRNAs encoding other chloride channels, such as CFTR and TTYH3, also did not match (Supplementary Materials Figure S1). Among the TTYH family, only TTYH1 and TTYH2 were well correlated with VRAC activity. As shown by Figure4, overexpression of TTYH1 or TTYH2 rescued VRAC currents in dKO cells. In addition, gene silencing of endogenous TTYH1 or TTYH2 decreased VRAC currents in HepG2 and LoVo cells, respectively. These data strongly indicate that both TTYH1 and TTYH2 act as VRACs in various cancer cells. VRAC currents are almost absent in cisplatin-resistant KCP-4 cells [11]. Consistent with this, we found that cisplatin-resistant R10 cells almost completely lacked VRAC currents (Figure1). These data suggest that reduced VRAC currents in cancer cells might be correlated with cisplatin resistance. We found that TTYH1 or TTYH2 could act as VRACs in cancer cells (SNU-601, HepG2, and LoVo cells), and that these channels were not expressed in MCF-7 cells (Figure5). Notably in this regard, LoVo cells expressing TTYH2 are much more sensitive to cisplatin (IC50, 0.8 µM) than MCF-7 cells (IC50, 15 µM), which poorly express TTYH1 and TTYH2 [21,22]. Therefore, it is worth investigating whether gene deficiency of TTYH1 or TTYH2 is associated with the cisplatin resistance in these cancer cells. In addition, TTYH1 and TTYH2 are upregulated in glioblastoma cells and colon cancer cells, respectively [23,24]. In these cancer cells, TTYH1 and TTYH2 regulate cell proliferation and migration, which can be effectively inhibited by silencing the corresponding gene [24]. Therefore, the functional roles of TTYH1- or TTYH2-mediated VRAC currents should be examined in these cancer cells. Interestingly, a recent report demonstrated that even though total LRRC8A expression in cisplatin-resistant A549 cells (A549CisR10) was increased compared with wild type A549 cells, taurine releasing activity of LRRC8A was reduced by the deficiency of LRRC8A surface expression [25]. Our present study also showed that mRNAs and proteins of LRRC8A were expressed in SNU-601 cells and R10 cells, but the VRAC currents were not affected by gene silencing of LRRC8A (Figures2 and4). Therefore, it is possible that the absence of LRRC8A-dependent VRAC currents could be mediated by the impairment of LRRC8A surface expression in SNU-601 cells. The regulatory mechanism of LRRC8A surface expression should be further investigated. In general, the functions and surface expression of ion channels are regulated by protein–protein interactions [26,27]. Interestingly, recent studies showed that TTYH1 can regulate Notch signaling in neural stem cells via protein–protein interaction with Rer1 (retention in endoplasmic reticulum sorting receptor 1), and surface expression of TTYH2 is suppressed by protein–protein interactions with β-COP [13,28]. In addition, the cellular level of TTYH2 is regulated by Nedd4-2–mediated ubiquitination [29]. These binding partners of TTYH1 or TTYH2 may be involved in the regulatory Cells 2019, 8, 562 12 of 14 mechanisms of VRACs in diverse normal tissues and cancer cells. Furthermore, LRRC8A and TMEM16A could be associated and cell swelling can stimulate the activities of LRRC8A and TMEM16A [30]. This finding implied that LRRC8A might interact with other ion channels. Therefore, we could not exclude the possibility of the functional interaction between TTYH channels and LRRC8A, and future studies are needed to test the possibility. Our results suggest that TTYH1 and TTYH2 can function as LRRC8A-independent VRACs. Further studies are needed to determine whether they are involved in other functions of VRACs, such as apoptosis and volume control in cancer cells. In particular, elucidation of the VRAC functions of TTYH1 as a VRAC could lead to improved treatment of brain tumors, which are currently associated with high mortality rates and limited therapeutic options.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/6/562/s1, Figure S1: Validations of LRRC8A shRNA, Figure S2: TTYH3 is expressed in a different pattern from TTYH1 and TTYH2, Table S1: Fold changes of the chloride channel genes from our microarray data. Author Contributions: Y.B. performed electrophysiological experiments on all cancer cells and dKO cells, and C.-H.C. and D.K. performed electrophysiological experiments on HEK cells and R10 cells. A.K. performed qRT-PCR, WB, and ICC for analysis of the microarray experiments. H.-G.J. and S.-S.K. generated and validated the shRNA constructs, and J.Y. screened the endogenous expression levels of TTYH1 and TTYH2 in various cancer cell lines. J.-Y.P. and E.M.H. designed and supervised the study, and wrote the manuscript. Funding: This study was financially supported by grant from the National Research Foundation (NRF-2017M3A9C4092979) awarded to JYP, and also grant from the Korea Institute of Science and Technology (2E29180) awarded to EMH. Conflicts of Interest: The authors declare no conflict of interest.

References

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