Overexpression of chloride channel-3 predicts unfavorable prognosis and promotes cellular invasion in gastric cancer

Introduction

Gastric cancer is one of the most aggressive and lethal malignancies in the world, especially in China.^1,2 Although the diagnosis, staging and treatment of gastric cancer have improved greatly, the prognosis remains poor, which is attributed to the high rates of tumor recurrence, including metastasis.^1,3 Therefore, it is of great value to identify new targets related to metastasis for individualized treatments and survival rates in patients with gastric cancer.

Chloride channel-3 (CLC-3), a member of the voltage-gated chloride channel superfamily,⁴ is involved in maintaining cell volume balance, regulating cell excitability, ion homeostasis, lysosomal acidification and transmembrane transportation.^5–7 Moreover, CLC-3 plays a critical role in regulation of cell proliferation, migration, invasion and apoptosis.^8–10 In addition to its critical roles in the physiological process, increasing evidence has found that CLC-3 is correlated with the malignant behavior of cancer cells. In nasopharyngeal carcinoma, CLC-3 was not only essential for cell proliferation and cell cycle progression but also contributed to cell migration and invasion.^11,12 In glioma, CLC-3 was reported to have an important role in the invasiveness of tumor cells through mediating resting chloride currents that participated in cell shrinkage process.^13,14 Additionally, CLC-3 has been found to contribute to tumorigenesis and tumor progression in osteosarcoma,¹⁵ endometrial cancer,¹⁶ prostate cancer¹⁷ and so on. A previous study showed the presence of CLC-3 protein in human gastric adenocarcinoma cell line;¹⁸ however, the clinical significance and biological function of CLC-3 in gastric cancer have not yet been elucidated.

In this study, we investigated the clinical significance, biological function and related molecular mechanism of CLC-3 in gastric cancer. CLC-3 is upregulated in gastric cancer tissues and cell lines, and elevated CLC-3 expression is associated with adverse clinicopathologic parameters and poor prognosis of gastric cancer patients. Functionally, suppression of CLC-3 prohibited, while CLC-3 overexpression promoted proliferation, migration and invasion of gastric cancer cells. Mechanically, knockdown of CLC-3 dramatically downregulated canonical transforming growth factor (TGF)-β/Smad signaling pathway, and supplement of TGF-β could partially rescue the suppressive effect of inhibition of CLC-3 on proliferation, migration and invasion of gastric cancer cells. Our data show the detailed role of CLC-3 in gastric cancer and further contribute to identify a candidate therapeutic target for the treatment of gastric cancer.

Materials and methods

Gene expression profile data from Gene Expression Omnibus (GEO)

We downloaded gastric cancer gene expression profile data GSE63089 and GSE56807 from open GEO database (http://www.ncbi.nlm.nih.gov/geo/), which contained 45 and 5 paired gastric cancer tissues and normal tissues, respectively. Bioinformatics software Qlucore Omics Explorer (QOE 3.1) (http://www.qlucore.com/) was used to analyze the CLC-3 mRNA expression level in primary gastric cancer tissues and paired normal tissues as previously described.¹⁹

Patients and tissue specimens

In this study, paraffin-embedded tissue samples from 353 patients with gastric cancer and corresponding 30 adjacent nontumor tissues were obtained from the first affiliated hospital, Sun Yat-sen University, Guangzhou, China, between 2001 and 2009. The patients were selected based on the following criteria: availability of resection tissue, follow-up data and should not have accepted preoperative radiation or chemotherapy. Patients who died from unknown causes or emergency were excluded from this study. This study was approved by the Institute Research Medical Ethics Committee of the First Affiliated Hospital, Sun Yat-sen University. A written informed consent was obtained from all the patients at the time of admission, with which the tissue, blood and other samples might be used for scientific research, but it did not relate to patient’s privacy.

Tissue microarray (TMA) construction

TMAs were constructed in accordance with a previously described method.²⁰ For each case, two cores taken from the selected tumor area or normal adjacent mucosa were used to construct the TMAs. Briefly, a hollow needle was utilized to punch and remove bipartite cylindrical tissue cores (1.0 mm in diameter) from selected donor tissue regions. Further, the punched tissue cores were inserted into a recipient paraffin block with a precisely spaced array pattern, using an automatic tissue arraying instrument (Beecher Instruments, Silver Spring, MD, USA).

Immunohistochemistry and evaluation

Immunohistochemical (IHC) staining was performed using a standard streptavidin–biotin–peroxidase complex method as described previously.²⁰ For antigen retrieval, tissue slides were heated in a microwave in 10 mM citrate buffer (pH 6.0) for 15 min. The slides were then incubated with rabbit polyclonal antibody against human CLC-3 (1:200, Abcam) overnight at 4°C in a moist chamber. A gastric tumor specimen was stained with PBS instead of primary antibody against CLC-3, and this was used as negative control. Protein expression levels of CLC-3 were evaluated by microscopic examination of the stained tissue slides. Brown cytoplasmic and/or membrane immunoreactivity for the CLC-3 protein was regarded as positive staining. CLC-3 expression level was evaluated by integrating the percentage of positive tumor cells and the intensity of positive staining. The intensity of staining was scored as follows: negative (score 0), weak (score 1), moderate (score 2) and strong (score 3). We scored the staining extent according to the percentage of positive stained cells in the field: negative (score 0), 0–25% (score 1), 26%–50% (score 2), 51%–75% (score 3) and 76%–100% (score 4). The sum of the intensity and extent scores was considered as the overall IHC score (values from 0 to 12). Then, we divided 353 patients with gastric cancer into two groups according to their CLC-3 expression: the high-expression group with a scoring index ≥8 (median score of CLC-3 expression; n=259) and the low-expression group with a scoring index <8 (n=94).

Cell lines and cultures

The human gastric cancer cell lines N87, MKN-45, BGC-823, MGC-803, SGC-7901 and the human gastric epithelial cell line (GES-1) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were grown in F-12k (American Type Culture Collection) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C with humidified 5% CO₂.

siRNA CLC-3, CLC-3 plasmid and transfection

Inhibition of CLC-3 expression in gastric cancer cells was determined using siRNA. Both nonspecific control siRNA (NC) and CLC-3 siRNA (SiCLC-3) were designed and synthesized by Shanghai GenePharma Company (Shanghai, China). The primers used were for SiCLC-3: sense 5′-CAAUGGAUUUCCUGUCAUATT-3′ and antisense 5′-UUCUCCGAACGUGUCACGUTT-3′, and for NC: sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′. When the cells were grown to 60% confluence, the SiCLC-3 or NC was transfected with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

For CLC-3 overexpression, ectopic CLC-3 coding sequence was amplified by polymerase chain reaction (PCR). The primer sequences were for SiCLC-3: sense 5′-CCAAGACCCCGCTTCAATAA-3′ and antisense 5′-CGAGTCCCGCAGATTAAAGA-3′, and for NC: sense 5′-TCAACTCTTTTGTGCCAGAGA-3′ and antisense 5′-CTCTACATTGGTGTTGAGCAATT-3′. The amplified product was cloned into the plasmid and confirmed by sequencing. The following transfection was performed in accordance with the manufacturer’s protocol.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from the gastric cancer cell lines using Trizol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Complementary DNA was prepared by using oligo^dT primers according to the protocol supplied with the Primer Script^™ RT Reagent (TaKaRa, Kyoto, Japan). Expression of CLC-3, TGF-β1 and TGF-β2 was determined by qRT-PCR using Power SYBR green PCR master mix (Applied Biosystems). The sequences of the PCR primers were as follows: CLC-3 sense 5′-TTGCCTACTATCACCACGAC-3′ and antisense 5′-GCATCTCCAACCCATTTACT-3′; TGF-β1 sense 5′-CCCACAACGAAATCTATGACAA-3′ and antisense 5′-AAGATAACCACTCTGGCGAGTC-3′; TGF-β2 sense 5′-ATTGCCCTCCTACAGACTTGAG-3′ and antisense 5′-CAGCACAGAAGTTGGCATTGTA-3′; GAPDH sense 5′-GCACCGTCAAGGCTGAGAAC-3′ and antisense 5′-TGGTGAAGACGCCAGTGGA-3′.

Cell proliferation assay

Cellular proliferation was measured by MTT and cell count assay. Briefly, for MTT assay, 1×10⁴ cells were seeded into a 96-well plate and incubated for 1, 2, 3 and 4 days. Twenty microliters of MTT (5 mg/mL; MP Biomedicals, Santa Ana, CA, USA) was added to each well and incubated for 4 h. The absorbance value (OD) of each well was measured at 570 nm. For cell count assay, 2×10⁵ cells from each group were plated into six-well culture plates in complete culture medium for 0, 1, 2, 3 and 4 days. Then, the cell number was determined in triplicate using a hemocytometer.

Cell migration and invasion assays

Transwell assay was used to assess the invasive and migratory potential of cells. For migration assay, briefly, 1×10⁵ cells were plated in serum-free media in the upper chamber of 24-well Transwell Chambers (Corning Incorporated, Corning, NY, USA), while media containing 10% fetal bovine serum were added to the lower chamber as a chemoattractant. After the cells were incubated for 48 h, crystal violet–stained cells adhering to the lower surface were counted. For cell invasion assay, the procedure was similar to the cell migration assay, except that the Transwell membranes were precoated with 0.25 mg/mL Matrigel (Corning Incorporated).

Western blot analysis

Cells were harvested and lysed with the radio immunoprecipitation assay buffer. Protein concentration was determined by the Bradford method with bovine serum albumin as the control. Equal amounts of tissue lysates (50 μg) were run by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred on a polyvinylidene difluoride membrane. The membrane was then blocked and incubated with primary antibodies against CLC-3 (1:1000, Abcam), TGF-β1 and TGF-β2 (1:400, Santa Cruz), Smad2 and Smad3 (1:5000, Abcam), phospho-Smad2 (1:1000, Abcam), phospho-Smad3 (1:2000, Abcam), Snail (1:1000, Abcam), ZEB1 (1:3000, Abcam), Slug (1:2000, Abcam), Twist1 (1:1500, Abcam) and GAPDH (1:2500, Abcam). Afterward, the membranes were washed three times, incubated with secondary antibody for 1 h at room temperature and visualized with enhanced chemiluminescence.

Statistical analysis

Statistical analysis was carried out with the SPSS statistical software package (SPSS Standard version 21.0, IBM Corporation) and GraphPad Prism 5. We assessed the association of CLC-3 protein expression with clinicopathologic features by the Chi-square test or Fisher’s exact test. For univariate survival analysis, Kaplan–Meier analysis was used. We utilized the log-rank test to compare different survival curves. The multivariate Cox proportional hazards regression model was used to assess the potential independent prognostic factors and 95% CI of hazard ratios. P value of <0.05 was considered statistically significant.

Results

CLC-3 is highly expressed in gastric cancer cell lines and tissues

To examine the expression level of CLC-3 in gastric cancer, we first downloaded two cohorts of gastric cancers, GSE63089 and GSE56807, from GEO database. Results from QOE 3.1 showed that CLC-3 mRNA level was significantly higher in gastric cancer tissues than in adjacent nontumor tissues in both GSE63089 and GSE56807 (P<0.05 and P<0.01, respectively; Figure 1A). Next, we detected the expression levels of CLC-3 mRNA and protein in gastric cancer cell lines using qRT-PCR and Western blot. As shown in Figure 1B, C, the expression of CLC-3 was elevated in MKN45, BGC-823, MGC-803 and SGC-7901 gastric cancer cell lines than that in the normal gastric epithelial cell line GES-1, both in mRNA and protein levels. Then, to further validate these findings, we performed IHC staining in TMAs including 353 gastric cancer tissues and 30 adjacent nontumor tissues. As expected, high expression of CLC-3 protein was detected in 259/353 (73.4%) primary gastric cancer tissues, while 25/30 (83.3%) adjacent nontumor mucosa showed only a low expression (P<0.01; Figure 2A). The expression of CLC-3 was mainly located in the cytoplasm and/or membrane, and in just a few cases (13/353) in the nucleus. Taken together, our results indicate that both mRNA and protein levels of CLC-3 are upregulated in gastric cancer.

Figure 1 CLC-3 is highly expressed in gastric cancer. Notes: (A) Heat map of QOE 3.1 software (http://www.qlucore.com/) represents the levels of CLC-3 mRNA expression of GSE63089 and GSE56807 datasets. The results show that CLC-3 was significantly higher in T than N (P<0.05 and P<0.01, respectively). (B)The relative mRNA levels of CLC-3 were detected by qRT-PCR in gastric cancer cell lines and GES-1. The data are presented as the mean±SD. (C) Western blot analysis of CLC-3 protein expression in gastric cancer cell lines and GES-1. (**P<0.01). GAPDH served as a loading control. Abbreviations: CLC-3, chloride channel-3; QOE, Qlucore Omics Explorer; qRT-PCR, quantitative real-time PCR; T, gastric cancer tumors; N, non-tumor tissues; GES-1, gastric epithelial cell line.

Figure 2 CLC-3 is elevated in gastric cancer tissues and predicts a poor prognosis. Notes: (A) Immunohistochemical staining revealed CLC-3 protein’s high expression (a, b) and low expression (c) in gastric cancer tissues, and low expression in adjacent non-tumor tissues (d). (a, scoring index=12; b, scoring index=8; c, d, scoring index=0). The lower panels display representative immunohistochemical staining patterns in selected zones with an enlarged view. Scale bars=100 μm; magnification of the upper images ×40, and the lower images ×100. (B) Survival curve for 353 gastric cancer patients according to CLC-3 protein expression status (log-rank test). Gastric cancer patients with a high expression of CLC-3 protein had a significantly shorter OS time than those patients with a low expression of CLC-3 (P=0.000). Abbreviations: CLC-3, chloride channel-3; OS, overall survival.

Overexpression of CLC-3 is correlated with adverse clinicopathologic parameters

To explore the potential clinical significance of CLC-3 in gastric cancer, the relationship between CLC-3 protein expression and clinicopathologic parameters was analyzed. High or low expression rates of CLC-3 protein in gastric cancer with respect to several standard clinicopathologic features are presented in Table 1. Overexpression of CLC-3 was positively associated with tumor size (P=0.005), lymph node metastasis (P=0.000) and TNM stage (P=0.000). However, we failed to discover any statistically significant relationship between the expression level of CLC-3 and other clinical parameters, such as gender, age at surgery and histologic type. These results demonstrate that CLC-3 might play a crucial role in gastric cancer progression.

Table 1 Correlation of CLC-3 expression with clinicopathologic parameters Notes: aChi-square test; bmedian age. Bold figures indicate P<0.05. Abbreviation: CLC-3, chloride channel-3.

In addition, nuclear staining of CLC-3 had no significant association with patients’ prognosis and malignant clinicopathologic parameters such as tumor size, lymph node metastasis and TNM stage (data not shown).

High CLC-3 expression predicts gastric cancer patients’ poor prognosis

To further examine the relationship between the expression of CLC-3 and the clinical prognosis of gastric cancer patients, we conducted survival analyses by univariate and multivariate Cox regression analysis. In univariate survival analysis, Kaplan–Meier survival curve and P value were manipulated by log-rank method. As shown in Figure 2B, gastric cancer patients with high expression of CLC-3 had a significantly shorter overall survival time than those patients with low expression of CLC-3 (P=0.00). The mean value of overall survival time was 34.22±1.96 months in patients with high levels of CLC-3, compared to 46.08±2.59 months in patients with low expression of CLC-3.

Multivariate Cox regression analysis was used to evaluate the potential prognostic significance of CLC-3 expression and other parameters. As shown in Table 2, high expression of CLC-3 was an independent prognostic factor for poor overall survival (hazard ratio 0.460, 95% CI 0.285–0.742; P=0.001), as well as histologic type, TNM stage and lymph node metastasis status (P=0.024, P=0.000 and P=0.000, respectively).

Table 2 Multivariate analysis on overall survival (Cox regression model) Notes: aHigh expression vs. low expression; bmale vs. female; c<57 vs. ≥57 years; d≥5 vs. <5 cm; eintestinal vs. diffuse; fstage I+II vs. stage III+IV; gabsent vs. present. Bold figures indicate P<0.05. Abbreviation: CLC-3, chloride channel-3.

Knockdown of CLC-3 inhibited gastric cancer cells’ proliferation, migration and invasion

In order to find out the biological function of CLC-3 in gastric cancer, we first suppressed the expression of CLC-3 in BGC-823 and SGC-7901 cells which expressed a relatively higher level of CLC-3. The decreased expression of CLC-3 was confirmed by Western blot (Figure S1). Then, MTT and cell count assay were employed to evaluate the proliferation. As shown in Figure 3A, MTT and cell count assay showed that downregulation of CLC-3 expression markedly abrogated gastric cancer cell viability or the number of gastric cancer cells, compared to that in the control group (P<0.01 and P<0.01, respectively). Similar to BGC-823 cells, knockdown of CLC-3 also inhibited proliferation of SGC-7901 cells (Figure 3B). Then, we assessed the effect of CLC-3 on gastric cancer cell invasion through Transwell assay. As shown in Figure 3C, regarding BGC-823 cells, the number of migrated and invasive cells in the SiCLC-3 group was notably decreased compared with that in the NC group. Likewise, decreased expression of CLC-3 could significantly inhibit cell migration and invasion in SGC-7901 cells (Figure 3D).

Figure 3 Knockdown of CLC-3 inhibits gastric cancer cells’ proliferation, migration and invasion. Notes: MTT (right panel) and cell count assay (left panel) indicate that reduced CLC-3 expression markedly abrogated cell viability or the number cells of (A) BGC-823 and (B) SGC-7901 cells compared with those of the control group (**P<0.01 vs. NC group). Experiments were performed in triplicate. Bars: SD. Transwell assay showed that inhibition of CLC-3 reduced the number of migrated and invasive (C) BGC-823 cells and (D) SGC-7901 cells compared with those of the control group (**P<0.01). Experiments were performed in triplicate. Bars: SD. Magnification ×100. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA; SiCLC-3, CLC-3 siRNA.

Elevated expression of CLC-3 promotes proliferation, migration and invasion of gastric cancer cells

To further confirm the role of CLC-3 in gastric cancer cells, we performed gain-of-function experiments through upregulating the expression of CLC-3 in N87 cells, which showed a lower level of CLC-3. The elevated expression of CLC-3 was validated by Western blot (Figure S2). As expected, MTT and cell count assay indicated that cell proliferation was notably enhanced by upregulating CLC-3 expression. Transwell assay revealed that overexpression of CLC-3 significantly increased the migratory and invasive abilities of N87 cells (Figure 4).

Figure 4 Overexpression of CLC-3 promotes gastric cancer cells’ proliferation, migration and invasion. Notes: (A) MTT and cell count assay indicated that cell proliferation in N87 cells was notably enhanced by upregulating CLC-3 expression. (B) Transwell assay revealed that overexpression of CLC-3 significantly increased migratory and invasive abilities of N87 cells. **P<0.01. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA.

TGF-β/Smad signaling pathway is involved in CLC-3–induced gastric cancer cell proliferation, migration and invasion

TGF-β signaling, which was related to cell proliferation, migration and invasion, was reported to be involved in CLC-3–mediated physiopathologic processes.^21,22 To determine whether TGF-βs were also necessary for the influence of CLC-3 on gastric cancer cell proliferation, migration and invasion, we first detected the effect of SiCLC-3 on TGF-βs expression in BGC-823 and SGC-7901 cells. As shown in Figure 5A, B, knockdown of CLC-3 significantly reduced the expression of TGF-β1 and TGF-β2 both in mRNA and protein levels. Moreover, canonical TGF-β/Smad signaling pathway related proteins, phospho-Smad2 and phospho-Smad3, were also decreased (Figure 5B). In addition, the downstream genes of TGF-β/Smad pathway, such as Snail, ZEB1, Slug, except for Twist1, were significantly decreased in gastric cancer cells when CLC-3 was knocked down (Figure 5C).

Figure 5 TGF-β/Smad signaling pathway is involved in CLC-3–induced gastric cancer cell migration and invasion. Notes: (A) qRT-PCR implies that inhibition of CLC-3 prohibited the expression of TGF-β1 and TGF-β2 mRNA in BGC-823 and SGC-7901 cells (**P<0.01). (B) Western blot analysis reveals that inhibition of CLC-3 suppressed the protein expression level of TGF-β1, TGF-β2, p-Smad2 and p-Smad3 (**P<0.01). GAPDH was used as an internal standard for protein loading. The values are presented as the mean±SD. (C) The downstream genes of TGF-β/Smad signaling pathway, such as Snail, ZEB1, Slug, except for Twist1, were significantly decreased in gastric cancer cells when CLC-3 was knocked down. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA; p-Smad2, phospho-Smad2; p-Smad3, phospho-Smad3; qRT-PCR, quantitative real-time PCR; SiCLC-3, CLC-3 siRNA.

Then, recombinant purified TGF-β was added to SiCLC-3 group at a concentration of 600 ng/mL. As expected, supplement of TGF-β effectively reversed the suppressed proliferation, migration and invasion induced by SiCLC-3 (Figure 6A, B). These data suggest that TGF-β/Smad signaling is required in CLC-3–induced gastric cancer proliferation, migration and invasion.

Figure 6 Supplement of TGF-β reverses the suppressed proliferation, migration and invasion induced by SiCLC-3. Notes: rTGF effectively reversed the suppressed (A) proliferation, (B) migration and invasion induced by inhibition of CLC-3 both in BGC-823 and SGC-7901 cells (*P<0.05; **P<0.01). Bars: SD. All of the experiments were performed in triplicate. Abbreviation: CLC-3, chloride channel-3; rTGF, recombinant purified TGF-β; NC, nonspecific control siRNA; SiCLC-3, CLC-3 siRNA; TGF, transforming growth factor.

Discussion

In this study, the clinical significance and biological function of CLC-3 in gastric cancer was investigated and it was demonstrated that 1) CLC-3 was upregulated in gastric cancer tissues and cell lines, both in mRNA and protein levels; 2) high expression of CLC-3 protein was correlated with adverse clinicopathologic parameters and predicted unfavorable prognosis; 3) CLC-3 expression was an independent negative prognostic factor for overall survival; 4) inhibition of CLC-3 expression suppressed, while overexpression promoted gastric cancer cell proliferation, migration and invasion in vitro and 5) knockdown of CLC-3 attenuated canonical TGF-β/Smad signaling pathway which was involved in CLC-3–induced gastric cancer cell proliferation, migration and invasion.

In gastric cancer, several chloride channels have been suggested to be critical for tumor progression and could serve as potential prognostic markers, for instance, chloride intracellular channel 1 expression was implied to accelerate the invasion and migration of gastric cancer cells and was associated with the TNM stage, perineural invasion, lymph node metastasis and the 5-year survival rate of gastric cancer patients²³ and Ca²⁺-activated chloride channel transmembrane protein 16A (TMEM16A) contributed to gastric cancer metastasis and was negatively associated with patients’ survival.²⁴ With regard to CLC-3, although several studies indicated that CLC-3 participated in cell proliferation, apoptosis, cell cycle and metastasis in many cancers,²⁵ the clinical significance of CLC-3 expression has not been well implicated. Xu et al²⁶ analyzed the prognostic value of CLC-3 expression in three tumors including lung adenocarcinoma, breast cancer and liver cancer and found that high expression of CLC-3 was an unfavorable prognostic marker for tumor patients. In our current study, we demonstrated that high CLC-3 expression was significantly associated with adverse clinicopathologic features such as tumor size, lymph node metastasis, TNM stage and poor prognosis. What is more, multivariate Cox regression analysis indicated that CLC-3 expression was an independent prognostic factor for gastric cancer patients. It seems that CLC-3 may be a putative oncogene in gastric cancer and may play a pivotal role in tumor progression.

CLC-3 has been reported to localize in the plasma membrane, cytoplasm and nuclei in normal and cancer cells. The functional role of CLC-3 was thought to be related to its subcellular location. For instance, membrane ClC-3 was a component and/or regulator of the volume-activated Cl− channel,²⁷ and intracellular vesicle CLC-3 could work as a Cl− channel to facilitate vesicle acidification.²⁸ On the other hand, the cellular distribution of CLC-3 could change with the process of cell activities or function. Mao et al²⁹ showed that CLC-3 could shuttle between the cytoplasm and the nucleus during different stages of the cell cycle. In our study, IHC staining could detect CLC-3 mainly in the cytoplasm and/or membrane and in just a few cases (13/353) in the nucleus. Unlike cytoplasm and/or membrane staining, nuclear staining has no association with prognosis and clinicopathologic features. Nevertheless, whether the effect of CLC-3 in gastric cancer cells is dependent on its subcellular localization and its related mechanisms needs to be further explored.

Tumor cells must undergo several morphologic changes in cell shape and volume during migration and invasion.³⁰ Chloride channels, especially volume-regulated chloride channels (VRCC), may play a critical role in tumor cell migration and invasion due to their ability to modulate cell volume through regulating regulatory volume decrease (RVD).^31–33 To date, considerable studies have found that CLC-3, a strong candidate of VRCC, is involved in the modulation of cell migration and invasion. Mao et al^12,34 reported that suppression of CLC-3 expression reduced cell migration in a concentration-dependent manner and inhibited the VRCC and RVD of nasopharyngeal carcinoma CNE-2Z cells. In gliomas, SiCLC-3 or the inhibitor (chlorotoxina) could partially suppress invasion of glioma STTG1 and U251-MG cells.³⁵ In endometrial cancer, CLC-3 could act as a VRCC to facilitate tumor migration and invasion through inducing RVD and mediating [Ca2+]i increase.¹⁶ These data strongly suggest that CLC-3 may regulate tumor cell migration and invasion by modulating the cell volume as a key molecular component of VRCC. Consistent with previous studies, our data showed that silencing of CLC-3 inhibits gastric cancer cell migration and invasion in vitro. However, the underlying mechanism was unclear. Jin et al¹⁸ demonstrated that CLC-3 might be the fundamental molecular entity responsible for VRCC and RVD in human gastric cancer AGS cells. Hence, it was supposed that CLC-3 might promote gastric cancer cell migration and invasion through regulating cell volume via acting as a functional entity of VRCC. Nevertheless, further studies should be performed to explore this.

TGF-β signaling regulates various cellular processes including proliferation, apoptosis, differentiation, cytokine secretion and so on.³⁶ Accumulating evidence shows that TGF-β signaling may play a vital role in the invasion and metastasis of different cancers, including gastric cancer.^37–39 Previous studies indicated that TGF-β signaling was involved in CLC-3–mediated physiopathologic processes. Wang et al²¹ found that CLC-3 chloride channel acted as a mechanically sensitive channel to regulate osteodifferentiation in osteoblasts through TGF-β signaling and others. Zheng et al²² indicated that CLC-3 deficiency attenuated cerebrovascular remodeling, possibly by the suppression of TGF-β signaling pathway in hypertension. In this study, we found that inhibition of CLC-3 significantly reduced the expression of two TGF-β isoforms, TGF-β1, TGF-β2, and phosphorylated Smad2/3, and downregulated the downstream genes of TGF-β/Smad signaling pathway such as Snail, ZEB1, Slug, except for Twist1. Moreover, supplement of TGF-β could partially rescue the suppressive effect of CLC-3 on gastric cancer proliferation, migration and invasion. These data suggest that canonical TGF-β/Smad signaling pathway is involved in CLC-3–induced gastric cancer cell migration and invasion. However, the mechanism of how CLC-3 regulates TGF-β signaling is unknown. Zheng et al²² showed that CLC-3 may be a key regulator for TGF-β/Smad3 cascade in cerebrovascular remodeling and CLC-3 deficiency could reverse deoxycorticosterone acetate-salt–induced altered expression of matrix metalloproteinase-2, which was reported as an activator of TGF-β.⁴⁰ On the contrary, Wang et al⁴¹ indicated that overexpression of CLC-3 inhibited the expression of TGF-β1 and promoted endosomal acidification. Strong acid or base was reported to be capable of activating the maximum TGF-β present in fibroblastic cell–conditioned medium.⁴² Hence, the authors speculated that CLC-3–induced TGF-β inhibition might be correlated with the changed intracellular microenvironment. The precise molecular mechanism underlying CLC-3–mediated aberrant activation of TGF-β signaling in gastric cancer cells needs further investigation.

Conclusion

Our study confirmed that CLC-3 was elevated in gastric cancer and CLC-3 expression was an independent negative prognostic potential marker. Silencing of CLC-3 inhibited, while overexpression promoted the proliferation, migration and invasion of gastric cancer cells through canonical TGF-β/Smad signaling pathway. These findings support the possibility of using CLC-3 as a novel therapeutic target for treating gastric cancer.

Acknowledgment

This study was supported by the National Natural Science Fund (No. 81272637 to Y He).

Disclosure

The authors report no conflicts of interest in this work.

References

1.		Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67(1):7–30.
2.		Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015. CA Cancer J Clin. 2016;66(2):115–132.
3.		Chou HH, Kuo CJ, Hsu JT, et al. Clinicopathologic study of node-negative advanced gastric cancer and analysis of factors predicting its recurrence and prognosis. Am J Surg. 2013;205(6):623–630.
4.		Hara-Chikuma M, Yang B, Sonawane ND, Sasaki S, Uchida S, Verkman AS. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J Biol Chem. 2005;280(2):1241–1247.
5.		Stauber T, Weinert S, Jentsch TJ. Cell biology and physiology of CLC chloride channels and transporters. Compr Physiol. 2012;2(3):1701–1744.
6.		Li X, Wang T, Zhao Z, Weinman SA. The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells. Am J Physiol Cell Physiol. 2002;282(6):C1483–C1491.
7.		Riazanski V, Deriy LV, Shevchenko PD, Le B, Gomez EA, Nelson DJ. Presynaptic CLC-3 determines quantal size of inhibitory transmission in the hippocampus. Nat Neurosci. 2011;14(4):487–494.
8.		Liang W, Huang L, Zhao D, et al. Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells. J Hypertens. 2014;32(2):318–330.
9.		Guan YT, Huang YQ, Wu JB, et al. Overexpression of chloride channel-3 is associated with the increased migration and invasion ability of ectopic endometrial cells from patients with endometriosis. Hum Reprod. 2016;31(5):986–998.
10.		Huang YY, Huang XQ, Zhao LY, et al. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice. Apoptosis. 2014;19(11):1559–1570.
11.		Xu B, Mao J, Wang L, et al. ClC-3 chloride channels are essential for cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells. Acta Biochim Biophys Sin (Shanghai). 2010;42(6):370–380.
12.		Mao J, Chen L, Xu B, et al. Suppression of ClC-3 channel expression reduces migration of nasopharyngeal carcinoma cells. Biochem Pharmacol. 2008;75(9):1706–1716.
13.		Lui VC, Lung SS, Pu JK, Hung KN, Leung GK. Invasion of human glioma cells is regulated by multiple chloride channels including ClC-3. Anticancer Res. 2010;30(11):4515–4524.
14.		Olsen ML, Schade S, Lyons SA, Amaral MD, Sontheimer H. Expression of voltage-gated chloride channels in human glioma cells. J Neurosci. 2003;23(13):5572–5582.
15.		Du S, Yang L. ClC-3 chloride channel modulates the proliferation and migration of osteosarcoma cells via AKT/GSK3beta signaling pathway. Int J Clin Exp Pathol. 2015;8(2):1622–1630.
16.		Li M, Wu DB, Wang J. Effects of volume-activated chloride channels on the invasion and migration of human endometrial cancer cells. Eur J Gynaecol Oncol. 2013;34(1):60–64.
17.		Lemonnier L, Shuba Y, Crepin A, et al. Bcl-2-dependent modulation of swelling-activated Cl- current and ClC-3 expression in human prostate cancer epithelial cells. Cancer Res. 2004;64(14):4841–4848.
18.		Jin NG, Kim JK, Yang DK, et al. Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells. Am J Physiol Gastrointest Liver Physiol. 2003;285(5): G938–G948.
19.		Liu F, He Y, Cao Q, Liu N, Zhang W. TBL1XR1 is highly expressed in gastric cancer and predicts poor prognosis. Dis Markers. 2016;2016:2436518.
20.		He XX, Ding L, Lin Y, Shu M, Wen JM, Xue L. Protein expression of HER2, 3, 4 in gastric cancer: correlation with clinical features and survival. J Clin Pathol. 2015;68(5):374–380.
21.		Wang H, Wang R, Wang Z, Liu Q, Mao Y, Duan X. ClC-3 chloride channel functions as a mechanically sensitive channel in osteoblasts. Biochem Cell Biol. 2015;93(6):558–565.
22.		Zheng LY, Li L, Ma MM, et al. Deficiency of volume-regulated ClC-3 chloride channel attenuates cerebrovascular remodelling in DOCA-salt hypertension. Cardiovasc Res. 2013;100(1):134–142.
23.		Chen CD, Wang CS, Huang YH, et al. Overexpression of CLIC1 in human gastric carcinoma and its clinicopathological significance. Proteomics. 2007;7(1):155–167.
24.		Liu F, Cao QH, Lu DJ, et al. TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-beta signaling. Oncotarget. 2015;6(13):11585–11599.
25.		Hong S, Bi M, Wang L, Kang Z, Ling L, Zhao C. CLC-3 channels in cancer (review). Oncol Rep. 2015;33(2):507–514.
26.		Xu B, Jin X, Min L, et al. Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival. Oncotarget. 2015;6(4):2434–2450.
27.		Hermoso M, Satterwhite CM, Andrade YN, et al. ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl- channels and volume regulation in HeLa cells and Xenopus laevis oocytes. J Biol Chem. 2002;277(42):40066–40074.
28.		Jentsch TJ, Maritzen T, Keating DJ, Zdebik AA, Thevenod F. ClC-3--a granular anion transporter involved in insulin secretion? Cell Metab. 2010;12(4):307–308, 309–310.
29.		Mao J, Li X, Chen W, et al. Cell cycle-dependent subcellular distribution of ClC-3 in HeLa cells. Histochem Cell Biol. 2012;137(6):763–776.
30.		Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. Curr Opin Cell Biol. 2005;17(5):559–564.
31.		Ransom CB, O’Neal JT, Sontheimer H. Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci. 2001;21(19):7674–7683.
32.		Schwab A. Function and spatial distribution of ion channels and transporters in cell migration. Am J Physiol Renal Physiol. 2001;280(5): F739–F747.
33.		Schwab A. Ion channels and transporters on the move. News Physiol Sci. 2001;16:29–33.
34.		Mao J, Chen L, Xu B, et al. Volume-activated chloride channels contribute to cell-cycle-dependent regulation of HeLa cell migration. Biochem Pharmacol. 2009;77(2):159–168.
35.		Lui VC, Lung SS, Pu JK, Hung KN, Leung GK. Invasion of human glioma cells is regulated by multiple chloride channels including ClC-3. Anticancer Res. 2010;30(11):4515–4524.
36.		Massague J. TGFbeta signalling in context. Nat Rev Mol Cell Biol. 2012;13(10):616–630.
37.		Miyazono K, Ehata S, Koinuma D. Tumor-promoting functions of transforming growth factor-beta in progression of cancer. Ups J Med Sci. 2012;117(2):143–152.
38.		Yokobori T, Nishiyama M. TGF-beta signaling in gastrointestinal cancers: progress in basic and clinical research. J Clin Med. 2017;6(1):11.
39.		Katz LH, Likhter M, Jogunoori W, Belkin M, Ohshiro K, Mishra L. TGF-beta signaling in liver and gastrointestinal cancers. Cancer Lett. 2016;379(2):166–172.
40.		Wang M, Zhao D, Spinetti G, et al. Matrix metalloproteinase 2 activation of transforming growth factor-beta1 (TGF-beta1) and TGF-beta1-type II receptor signaling within the aged arterial wall. Arterioscler Thromb Vasc Biol. 2006;26(7):1503–1509.
41.		Wang H, Mao Y, Zhang B, et al. Chloride channel ClC-3 promotion of osteogenic differentiation through Runx2. J Cell Biochem. 2010;111(1):49–58.
42.		Lyons RM, Keski-Oja J, Moses HL. Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium. J Cell Biol. 1988;106(5):1659–1665.

Supplementary materials

Figure S1 Confirmation of decreased expression of CLC-3 in gastric cancer cells. Notes: Western blot assay showed that SiCLC-3 suppressed CLC-3 protein expression significantly in BGC-823 and SGC-7901 cells. GAPDH acted as a loading control. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA; SiCLC-3, CLC-3 siRNA.

Figure S2 Confirmation of elevated expression of CLC-3 in gastric cancer cells. Notes: Western blot assay showed that CLC-3 plasmid upregulated CLC-3 protein expression significantly in N87 cells. GAPDH acted as a loading control. Abbreviations: CLC-3, chloride channel-3; NC, nonspecific control siRNA.