Sodium Channel Subunit SCNN1B Suppresses Gastric Cancer Growth and Metastasis Via GRP78 Degradation

Author Manuscript Published OnlineFirst on February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-1595 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Sodium channel subunit SCNN1B suppresses gastric cancer growth and metastasis via GRP78 degradation

Yun Qian1,2*, Chi Chun Wong1*, Jiaying Xu1, Huarong Chen1, Yanquan Zhang1, Wei Kang1,3, Hua Wang4, Li Zhang1, Weilin Li1, Eagle SH Chu1, Minnie Y.Y. Go1, Philip WY Chiu5, Enders KW Ng5, Francis KL Chan1, Joseph JY Sung1, Jianmin Si2, Jun Yu1

1Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China 2Department of Gastroenterology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China; Institute of Gastroenterology, Zhejiang University, Hangzhou, China 3Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China 4School of Biomedical Science, The Chinese University of Hong Kong, Hong Kong, China 5Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China *These authors contributed equally

Running title: SCNN1B as a novel tumor suppressor

Keywords: Gastric cancer, SCNN1B, tumor suppressor, DNA methylation, GRP78

Financial support: This project was supported by research funds from RGC-GRF (14114615 and 766613) from Hong Kong; Shenzhen Municipal Science and Technology R & D fund (JCYJ20130401151108652) and Shenzhen Virtual University Park Support Scheme to CUHK Shenzhen Research Institute; National 1

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 15, 2017; DOI: 10.1158/0008-5472.CAN-16-1595 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Natural Science Foundation of China (NSFC) (81502064); Direct grant for Research 2013/2014, CUHK (4054100).

Correspondence to: Professor Jun Yu, MD, PhD. Institute of Digestive Disease, Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. Phone: (852) 3763 6099; Fax: (852) 2144 5330; Email: [email protected]

Conflict of interest: The authors have no conflict of interest.

Word count: 4931 Total number of Figures and Tables: 7

Abstract

There remains a paucity of functional biomarkers in gastric cancer. Here we report the identification of the sodium channel subunit SCNN1B as a candidate biomarker in gastric cancer (GC). SCNN1B mRNA expression was silenced commonly by promoter hypermethylation in GC cell lines and primary tumor tissues. Tissue microarray analysis revealed that high expression of SCNN1B was an independent prognostic factor for longer survival in GC patients, especially those with late-stage disease. Functional studies demonstrated that SCNN1B overexpression was sufficient to suppress multiple features of cancer cell pathophysiology in vitro and in vivo. Mechanistic investigations revealed that SCNN1B interacted with the endoplasmic reticulum chaperone GRP78 and induced its degradation via polyubiquitination, triggering the unfold protein response (UPR) via activation of PERK, ATF4, XBP1s and CHOP and leading in turn to caspase-dependent apoptosis. Accordingly, SCNN1B sensitized GC cells to the UPR-inducing drug tunicamycin. GRP78 overexpression abolished the inhibitory effect of SCNN1B on cell growth and migration, while GRP78 silencing aggravated growth inhibition by SCNN1B. In summary, our results identify SCNN1B as a tumor suppressor function that triggers UPR in GC cells, with implications for its potential clinical applications as a survival biomarker in GC patients.

Introduction

Gastric cancer (GC) is one of the most common human cancers. Despite improvements in the surveillance and treatment of GC, it remains a devastating disease with poor prognosis (1). Epigenetic dysregulation plays an important role in gastric carcinogenesis. Previous studies have shown that the inactivation of tumor suppressor genes by promoter DNA methylation contributes to the pathogenesis of human GC (2-8). To unveil novel tumor suppressor genes that are silenced by epigenetic mechanisms in GC, we used genome-wide methylation array (Infinium Human Methylation 450K) to comprehensively profile CpG site methylation in five GC cell lines (AGS, HGC27, MGC803, MKN1 and MKN45), an immortalized human gastric epithelial cell GES1 and two normal gastric tissue samples. Using this approach, we identified SCNN1B as a novel gene that is highly methylated in human GC, whose potential role in GC development is largely unknown.

SCNN1B is located on chromosome 16p12.2 and it encodes β-subunit of the epithelial sodium channel (ENaC). SCNN1B is a part of a multi-protein complex consisting of three subunits (α, β and γ) that controls fluid and electrolyte transport across epithelia in diverse organs. SCNN1B is classified as a membrane channel, but accumulating evidence also indicates that ENaC subunits, including SCNN1B, participate in cellular differentiation (9-11). SCNN1A, which encodes the α-subunit of ENaC, has been shown to be silenced by promoter methylation in neuroblastoma and breast cancer (12, 13). However, the functional importance of SCNN1B in human cancer remains unexplored. In this study, we identified frequent silencing of SCNN1B in human GC, which was associated with promoter methylation. We demonstrated a significant correlation between the silence of SCNN1B protein expression and poor disease-specific survival of GC patients. We revealed that SCNN1B suppresses GC growth by inducing apoptosis and cell cycle arrest and inhibiting metastasis abilities. The tumor suppressive effect of SCNN1B was found to be mediated via: 1) the direct interaction with GRP78, a chaperone with oncogenic properties; 2) the reduction of 4

GRP78 protein by inducing its polyubiquitination and proteasome-mediated degradation; and 3) the induction of the UPR response, which activates PERK, ATF4, XBP1s and CHOP, leading to caspase-dependent apoptosis and cell cycle arrest. Moreover, Tissue microarray (TMA) analysis of 245 GC patients revealed that high SCNN1B expression is an independent prognostic factor that predicts better survival of GC patients.

Materials and Methods

Cell culture Sixteen GC cell lines and a normal gastric epithelial cell line were used in this study: AGS, KATOIII, MKN45 and NCI-N87 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD); MKN1, MKN74, SNU1, SNU638, SNU719 and YCC10 cells were obtained from the Korean Cell Line Bank (Seoul, Korea); MKN7 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan); BGC823, HGC27, MGC803, SGC7901 and normal gastric epithelial cell line GES1 were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were purchased between 2014 and 2015, and routinely cultured in DMEM containing 10% FBS and penicillin-streptomycin.

SCNN1B ectopic expression and knockdown The full length ORF of SCNN1B was cloned into pcDNA3.1, pCMV4-FLAG and pEGFP-N1 vectors. Transfection was performed with Lipofectamine 2000 (Life Technologies, Carlsbad, CA). Cell lines stably expressing SCNN1B were obtained after selection with neomycin (G418, Life Technologies) for at least 2 weeks. SCNN1B siRNA were purchased from Riobio Co. Ltd (Guangzhou, China) and transfected into MKN1 and NCI-N87 cells using Lipofectamine 2000.

Colony formation and cell growth curve assays

Cells were plated in 6-well plates at 1000 cells per well in complete DMEM. Medium was changed every 3 to 4 days. At the end point, cells were stained with 0.1% crystal violet and the number of colonies consisting of > 50 cells was counted. Cell growth curve was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO).

Apoptosis and cell cycle analysis Cells were plated in 12-well plates and serum-starved overnight. Annexin V-PE/7-aminoactinomycin D (7-AAD) staining kit (BD Biosciences, San Jose, CA) was used to determine cell apoptosis. For cell cycle, cells were serum-starved for 24 h and then stimulated with complete medium for 4 to 12h. Cell cycle distribution was then assessed by flow cytometry after staining with propidium iodide (Life Technologies).

Wound healing assay Confluent cultures in 6-well plates were scratched with sterile P-200 pipette tips, washed and cultured in DMEM containing 2% FBS. Cells were photographed after 0, 12, 24, and 48 h, respectively. Wound closure (%) was evaluated by the TScratch software.

Invasion assay Cell invasion was determined using BD BioCoat Matrigel Invasion Chamber (BD Biosciences). Cells (5x104/well) were seeded onto the upper chamber in serum-free DMEM. Complete DMEM (supplemented with 10% FBS) was added to the lower chamber as a chemo-attractant. After 48h, cells that have invaded through the membrane were stained with 0.1% crystal violet and counted.

Adhesion assay Cells (0.5-1x105/well) were seeded onto 96-well plates. After 30 and 60 min, the medium was aspirated, then the cells were washed with PBS and stained with 0.1% 6

crystal violet. The crystal violet was dissolved in 10% acetic acid overnight and absorbance was measured at 540nm.

Immunofluorescence Cells were seeded onto coverslips in a 6-well plate and transfected with GFP-tagged SCNN1B and Myc-tagged GRP78. At 24 h after transfection, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, blocked in 5% bovine serum albumin in PBS, and incubated with anti-Myc (1:4000 dilution) overnight at 4°C, followed by anti-mouse IgG secondary antibody conjugated with Alexa Fluor 594 (1:400 dilution, Yeasen, Shanghai, China) in the dark for 1 h. Cells were then mounted with ProLong® Gold Antifade Mountant with DAPI (Life technologies, Carlsbad, CA). Images were captured in a Carl Zeiss LSM 780 confocal laser-scanning microscope (Carl Zeiss AG, Oberkochen, Germany).

Human samples Paired primary gastric tumours and adjacent normal gastric tissues were collected immediately after surgical resection at the Prince of Wales Hospital. The specimens were snap-frozen in liquid nitrogen and stored at –80°C; and were also fixed in 10% formalin and embedded in paraffin for routine histologic examination. Biopsies from 3 cases of normal mucosa obtained during gastroscopy were recruited as healthy controls, which were confirmed by an experienced pathologist at the Prince of Wales Hospital. All patients gave informed consent, and the study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong.

TMA assay TMA was generated from formalin-fixed, paraffin-embedded archive tissues of 245 patients with GC prior to radiotherapy/chemotherapy, which were collected at the Prince of Wales Hospital, Hong Kong (14), with a median follow-up time of 40.8 months. All subjects provided informed consent for obtaining the specimens. TMA was stained with a commercially available anti-SCNN1B antibody (HPA015612, 7

Sigma-Aldrich, St Louis, MO). Anti-SCNN1B antibody (HPA015612) was confirmed by antibody specificity analysis with protein arrays, with single peak corresponding to interaction only with its own antigen. Cytoplasmic expression of SCNN1B was assessed by H-score. The proportion score was in the light of proportion of cancer cells with positive cytoplasmic staining (0, no positive staining; 1, in 10% or fewer cells; 2, in between 10% and 25% cells, 3, in between 25% and 50% cells,; 4, in more than 50% cells). The intensity score was assigned for the average intensity of cancer cells with positive staining (0, none; 1, weak; 2, intermediate; 3, strong). The immunohistochemistry (IHC) score of SCNN1B was calculated by the following formula: IHC score = proportional score (0 to 4) × intensity score (0 to 3), ranging from 0 to 12. Finally, the cytoplasmic expression of SCNN1B in GC tissue was divided into 3 groups according to IHC score (low, ≤3; intermediate, 4-6; high, 7-12). The results were scored independently by two pathologists and the average of the 2 values was taken.

Co-immunoprecipitation (co-IP)-Mass spectrometry Cells transiently transfected with SCNN1B-FLAG or empty vector were lysed with ice-cold RIPA lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS, pH 8.0) supplemented with protease inhibitors (Roche, Indianapolis, IN). Total proteins were immunoprecipitated using 2 µg of anti-Flag (F1804, Sigma-Aldrich) and bind to 40 µL of Protein G-Agarose (Santa Cruz Biotechnology, Dallas, Texas). After washing 5 times with RIPA buffer, bound proteins were eluted with loading buffer, separated by SDS-PAGE, and visualized by silver staining. Protein bands of interest in-gel were digested, and subjected to liquid chromatography-tandem mass spectrometry (ABI4800 MALDI TOF/TOF, Applied Biosystems, Foster City, CA). The MS fragment spectra were analyzed using Mascot software (Matrix Science, Boston, MA). To confirm the interaction of SCNN1B with GRP78, immune complexes were precipitated by anti-Flag and analyzed by Western blot using anti-Flag and anti-GRP78 (Sc-13968, Santa Cruz).

Ubiquitination assay AGS cells stably transfected with SCNN1B expression vector or empty vector were lysed with RIPA buffer supplemented with protease inhibitors. Immunoprecipitation was performed using anti-GRP78 or control IgG, respectively. Immunoprecipitated proteins were analyzed by Western blot using anti-ubiquitin (3936, Cell Signalling, Danvers, MA).

Subcutaneous xenografts model BGC823 (1x107 cells/0.1ml PBS) and MKN45 (1x106 cells/0.1ml PBS) cells stably expressing the control vector or SCNN1B were injected subcutaneously into the left and right dorsal flank of 4-6 weeks old female Balb/c nude mice (n=6/group), respectively. Tumor size was measured every 2 days for 2-3 weeks using a digital calliper. Tumor volume (V) was estimated by measuring the longest diameter (L) and shortest diameter (W) of the tumor and calculated by formula V = 0.5 × L × W2. At the end point, tumours were harvested and weighted. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.

Statistical analysis All the results were expressed as mean ± S.E.M. (continuous variables) or described as frequency and percentage (categorical data). To compare the difference between two groups, Independent Sample t-test or Mann-Whitney U test was used. The difference between growth rates was determined by ANOVA with repeated-measures analysis of variances. The Pearson chi-square test or Fisher’s exact test was used for analysis of the associations between patient clinicopathological characteristics and SCNN1B expression. Kaplan-Meier analysis and log-rank test were performed to evaluate the association between SCNN1B expression and disease-specific survival. COX proportion hazard regression model was performed to assess the prognostic value of SCNN1B expression. All the statistical analyses were performed using

GraphPad Prism, version 6.0 (GraphPad Software, San Diego, CA) or SPSS, version 20.0 (SPSS Inc, Chicago, IL). P<0.05 was considered statistically significant.

Results

Genome-wide methylation analysis identified SCNN1B promoter is densely methylated in human GC. Using the Infinium Human Methylation 450K array, we interrogated genome wide CpG methylation in five human GC cells (AGS, HGC27, MGC803, MKN1 and MKN45) as compared to normal gastric epithelial cell line GES1 and normal gastric tissues (Figure 1A). Using stringent criteria, we identified SCNN1B to be preferentially methylated at its promoter in GC (Figure 1B).

SCNN1B is silenced in GC cell lines and primary GC by promoter methylation We initially examined SCNN1B mRNA expression in human normal tissues, and found that SCNN1B was widely expressed in most human normal tissues with strong expression in the stomach (Figure S1). On the other hand, SCNN1B mRNA expression was silenced in 13 out of 16 (81.3%) GC cell lines (Figure 1C), only MKN1, MKN7 and NCI-N87 cells lines expressed significant levels of SCNN1B mRNA (Figure 1C & S2). To determine the role of promoter methylation in silencing of SCNN1B, we evaluated its promoter methylation by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS). MSP analysis revealed dense SCNN1B promoter methylation in all GC cell lines with silenced SCNN1B expression (Figure 1C). BGS analysis of 38 CpG sites in SCNN1B promoter and the first exon showed dense methylation (average methylation >50%) in the SCNN1B-silenced GC cell lines examined, but not in SCNN1B-expressing MKN1 cells and normal gastric tissues (Figure 1D and S3). To test if promoter methylation directly mediates the silencing of SCNN1B, six GC cell lines with silenced SCNN1B expression were treated with DNA methyltransferase inhibitor, 5-Aza-2’-deoxycytidine (5-Aza). 5-Aza restored SCNN1B expression in all six cell lines, indicating that promoter 10

methylation contributes to the transcriptional silencing of SCNN1B (Figure 1E). In addition, treatment with 5-Aza plus histone deacetylase inhibitor trichostatin A (TSA) could fully restore SCNN1B expression in MKN45 cells with moderate promoter methylation (Figure 1E).

We evaluated mRNA and protein expression of SCNN1B in gastric tissues from 74 primary GC patients. SCNN1B mRNA was significantly down-regulated in GC as compared to paired adjacent normal gastric tissues (P<0.0001) (Figure 2A and 2B). SCNN1B mRNA expression was also down-regulated in GC in the TCGA cohort (n=34, P<0.0001) (Figure 2B). Western blot and immunohistochemistry confirmed the reduced expression of SCNN1B in GC as compared to adjacent normal tissues (n=10, P<0.001) (Figure 2A and 2B). We next examined the methylation status of SCNN1B in primary GC. MSP and BGS analysis demonstrated that SCNN1B promoter methylation was significantly higher in GC as compared to adjacent normal tissues (Figure 2A and 2C). None of the normal gastric biopsies showed SCNN1B promoter methylation. These data implied that SCNN1B is silenced by promoter methylation in GC. Consistent with our data, analysis of the TCGA dataset revealed an inverse correlation between SCNN1B mRNA expression and promoter methylation in GC (P<0.001) (Figure 2D).

SCNN1B expression is an independent predictor of favourable outcome in GC patients To evaluate the association of SCNN1B expression with clinicopathological features and clinical outcomes, we assessed the SCNN1B protein expression in GC utilizing a GC TMA (n=245). SCNN1B cytoplasmic expression showed a significant correlation with TNM stage (P<0.001) and lymphatic metastasis (P=0.036), but had no correlation with age, gender, H pylori infection, histological Lauren classification or tumor grade (Table S1). In univariate Cox regression analysis, an intermediate or high cytoplasmic SCNN1B score was associated with better disease specific survival (Intermediate: HR: 0.482, 95% CI: 0.320 to 0.726, P<0.001; High: HR: 0.247, 95% 11

CI: 0.091 to 0.674, P=0.006). Apart from SCNN1B expression, age (P=0.048), histological Lauren classification (P<0.001), tumor grade (P=0.024), and TNM stage (P<0.001) were also correlated with survival by univariate analysis. After adjustment for potential confounding factors such as age, gender, histological Lauren classification, tumor grade and TNM stage, SCNN1B expression was found to be an independent prognostic factor for disease-specific survival (Intermediate: HR: 0.547, 95% CI: 0.360 to 0.829, P=0.005; High: HR: 0.353, 95% CI: 0.128 to 0.971, P=0.044) by multivariate Cox proportional hazards regression analysis (Table S2). As shown by Kaplan-Meier curves, GC patients with intermediate or high SCNN1B protein expression had significantly longer survival (P<0.001) (Figure 2E). Further stratification of the TMA cohort into early stage (TNM stage I/II) and late stage (TNM stage III/IV) revealed that intermediate or high protein expression of SCNN1B was associated with better survival in late stage GC (P=0.011) (Figure 2F). Analysis of another two independent GC cohorts (GSE62254 and GSE14210) also showed that high SCNN1B mRNA expression was associated with better survival in late stage GC (Figure S4, and Table S3-S4). These results indicate that high SCNN1B expression predicts a favourable prognosis in patients with GC.

SCNN1B suppresses GC cell growth through the induction of apoptosis and cell cycle arrest The frequent silencing of SCNN1B in GC and its association with patient survival prompted us to hypothesize that SCNN1B functions as a tumor suppressor. To this end, we generated four GC cell lines (AGS, BGC823, MGC803 and MKN45) with stable SCNN1B expression. Ectopic expression of SCNN1B was validated by RT-PCR and Western blot (Figure 3A), which was comparable to that of normal gastric tissues (Figure S5). SCNN1B overexpression suppressed colony formation ability by 55-80% as compared to empty vector transfected cells in all four GC cell lines (Figure 3B, P<0.01). Consistently, cell growth curve assay revealed that ectopic SCNN1B expression inhibited viability in these cell lines (Figure 3C, P<0.001).

To determine the cytokinetic effect of SCNN1B on GC cells, we analyzed apoptosis and cell cycle distribution by flow cytometry. Overexpression of SCNN1B led to a significant increase in the total apoptotic cell population in AGS (P<0.05), BGC823 (P<0.01), MGC803 (P<0.001) and MKN45 (P<0.001) cells, as determined by Annexin V-PE/7-AAD dual staining (Figure 3D). Induction of apoptosis by SCNN1B was confirmed by the elevated expression of key apoptosis markers such as cleaved forms of caspase-9, caspase-8, caspase-7 and poly ADP ribose polymerase (PARP), as determined by Western blot (Figure 3D). We also observed an increased accumulation

of GC cells in the G1 phase (P<0.05) and a reduction of S phase population (P<0.05)

following ectopic SCNN1B expression (Figure 3E). Consistent with G1 arrest, we

Kip1 found that SCNN1B increased the expression of G1 phase gatekeepers p27 and p53; whilst reducing expression of cyclin D1 and CDK2, both of which are important for

G1 progression (Figure 3E). Next, we performed loss of function experiments using two independent SCNN1B targeted siRNA to knockdown endogenous SCNN1B in MKN1 and NCI-N87 cells (Figure 3F and S6A). The knockdown of SCNN1B increased colony formation ability (P<0.001) and promoted cell cycle progression in MKN1 and NCI-N87 cells (P<0.01) (Figure 3F, S6B and S6C). These data indicate that SCNN1B suppresses GC cell proliferation.

SCNN1B regulates GC cell migration, invasion and adhesion In light of the association between SCNN1B expression and metastasis of GC patients, we next ask whether SCNN1B has an effect on cell migration, adhesion and invasion. SCNN1B overexpression markedly suppressed cell migration in AGS, BGC823, MGC803 and MKN45 cell lines by wound healing assay. Quantitative analysis demonstrated a significant impairment in wound closure at different time points (P<0.001) in SCNN1B-overexpressing cells, thereby suggesting that SCNN1B negatively regulates cell migration (Figure 4A). SCNN1B also promoted cell adhesion in all four GC cell lines (Figure 4B). In addition, Matrigel invasion assay revealed that ectopic expression of SCNN1B suppressed cell invasion in AGS, BGC823 and MGC803 cells by over 50% (P<0.001) (Figure 4C). Conversely, siRNA 13

mediated SCNN1B silencing in MKN1 and NCI-N87 cells resulted in enhanced wound closure (P<0.001), but decreased cell adhesion (P<0.05) as compared to control (Figure 4D, 4E, S6D and S6E). Thus, SCNN1B reduces the metastatic ability of GC cells by inhibiting cell migration and invasion, whilst promoting cell adhesion.

Ectopic SCNN1B expression inhibits tumorigenicity in nude mice In light of our in vitro results, we evaluated the impact of ectopic SCNN1B expression in the nude mice tumorigenicity assay. MKN45 and BGC823 cell lines with stable expression of empty vector or SCNN1B were injected into the left and right flanks of nude mice, respectively. As shown in Figure 5, tumor growth was significantly slower in mice injected with MKN45-SCNN1B cells than those with MKN45-emtpy vector cells (P<0.01) (Figure 5A) and in mice with BGC823-SCNN1B cells than those with BGC823-emtpy vector cells (P<0.01) (Figure 5B). The average tumor weight at sacrifice was significantly lower in MKN45-SCNN1B mice (P<0.05) (Figure 5A) and in BGC823-SCNN1B mice (P<0.05) (Figure 5B) as compared to their corresponding control mice. Ectopic SCNN1B expression in the tumor xenografts of MKN45-SCNN1B and BGC823-SCNN1B were confirmed by RT-PCR and Western blot (Figure 5A, 5B and S7). Ki-67 staining revealed a significant reduction in cell proliferation in MKN45 tumors expressing SCNN1B (P<0.05) (Figure 5A). These results supported a tumor suppressive role for SCNN1B.

SCNN1B interacts with GRP78 and mediates GRP78 protein degradation via polyubiquitination To further elucidate the molecular mechanism underlying the tumor suppressive effect of SCNN1B, we performed co-IP of Flag-tagged SCNN1B in HEK293 cells, followed by mass spectrometry in order to identify its binding partners (Figure 6A). The 78kDa glucose-regulated protein (GRP78) was identified as a potential interacting partner for SCNN1B. GRP78 is a stress-inducible chaperone that normally resides in endoplasmic reticulum; however, recent advances have shown that GRP78 plays an oncogenic role in cancer via supporting cell proliferation, invasion and metastasis, 14

and inhibition of apoptosis (15-17). We validated the interaction between SCNN1B and GRP78 in AGS and BGC823 cells (Figure 6A), in which GRP78 was co-immunoprecipitated by Flag-tagged SCNN1B in both cell lines. We next evaluated co-localization of SCNN1B and GRP78 by immunofluorescence in AGS, BGC823 and MKN45 cell lines (Figure 6B). Confocal microscopy images showed that GFP-tagged SCNN1B was found in the membrane and cytoplasm; whereas Myc-tagged GRP78 was expressed in membrane and cytoplasm. Co-localization of SCNN1B and GRP78 was observed mainly the cytoplasm. These results indicate that SCNN1B is a binding partner of GRP78.

We next assessed the interplay between SCNN1B and GRP78. GRP78 mRNA levels were not altered following the ectopic expression of SCNN1B (Figure S8) On the other hand, SCNN1B expression strongly attenuated expression of GRP78 at protein level in GC cell lines (Figure 6C). Moreover, SCNN1B decreased the expression of GRP78 in the cytoplasm and membrane that was consistent with their co-localization, thus implying that SCNN1B might regulate GRP78 via protein degradation (Figure 6C).

Protein degradation in eukaryotic cells is mediated by two major pathways: ubiquitin-proteasome and autophagy-lysosomal pathways. To pinpoint the mechanism of SCNN1B-induced GRP78 degradation, we treated SCNN1B overexpressing AGS cells with inhibitors of the proteasome (MG132) and lysosome (chloroquine). MG132, but not chloroquine, restored GRP78 protein levels in SCNN1B-overexpressing AGS cells (Figure 6D), implying that the ubiquitin-proteasome pathway was involved in the degradation of GRP78. To validate this, we examined polyubiquitination of GRP78 with or without ectopic SCNN1B expression (Figure 6D). Indeed, SCNN1B increased polyubiquitination of GRP78. Collectively, these data indicated that SCNN1B directly interacts with GRP78 and mediates GRP78 degradation via ubiquitination.

Down-regulation of GRP78 by SCNN1B induces cell death via UPR GRP78 is a central regulator of the unfolded protein response (UPR) by sequestration of three canonical branches, PERK-eIF2α-ATF4, IRE1α-XBP1s and ATF6 pathways. Given that GRP78 is down-regulated by SCNN1B, we evaluated the activation of the three UPR signalling pathways. Increased expression of PERK, XBP1s and ATF4 were demonstrated in SCNN1B overexpressing GC cell lines by Western blot (Figure 6E). In addition, nuclear abundance of ATF4 and XBP1s was simultaneously induced in SCNN1B-expressing GC cells, suggesting that these transcription factors were activated (Figure 6E). We also observed the up-regulation of C/EBP homologous protein (CHOP), a key mediator of UPR mediated apoptotic pathway, in SCNN1B overexpressing GC cell lines (Figure 6E). To test if induction of UPR plays an important role in tumor suppressive effect of SCNN1B, we co-incubated control and SCNN1B expressing GC cells with the UPR stress inducer tunicamycin. We found that overexpression of SCNN1B sensitized AGS and MKN45 cells to the cytotoxic

effect of tunicamycin as compared to controls. IC50 values of tunicamycin in AGS-empty vector and AGS-SCNN1B cells were 368 and 234 ng/mL, respectively. A

similar trend was observed in the MKN45 cell line, where empty vector cells (IC50:

1175 ng/mL) were less sensitive than SCNN1B-expressing cells (IC50: 886 ng/mL) to tunicamycin (Figure 6F). Collectively, induction of UPR plays an important role in tumor suppressive function of SCNN1B in GC.

The tumor suppressive effect of SCNN1B is dependent on down-regulation of GRP78. Given that SCNN1B abrogated GRP78 expression through polyubiquitination, we next conducted rescue experiments in which we co-transfected the control and SCNN1B expressing GC cells with empty vector or GRP78. We first evaluated the effect of ectopic GRP78 expression on cell proliferation. As shown in Figure 7A, ectopic expression of GRP78 restored GRP78 protein levels in SCNN1B-overexpressing cells. Moreover, colony formation assay showed that GRP78 restored the number of cell colonies in SCNN1B-expressing cells to baseline levels in AGS cells (P<0.01), whilst GRP78 overexpression did not promote colony formation 16

in control cells (Figure 7A). This indicated that growth suppressive effect of SCNN1B is mediated by down-regulation of GRP78. We next investigated effect of GRP78 overexpression on metastatic capacity of SCNN1B overexpressing AGS cells. While control AGS cells had comparable wound closure rate irrespective of GRP78 expression, GRP78 promoted wound closure in SCNN1B overexpressing AGS cells (P<0.001) (Figure 7B), implying that SCNN1B-mediated degradation of GRP78 contributed to its anti-metastatic effect in GC cells. In contrast, siRNA-mediated knockdown of GRP78 was additive with ectopic SCNN1B expression to suppress GRP78 expression and inhibit AGS cell growth as compared with empty vector control (Figure 7C). These findings pointed to a pivotal role of GRP78 modulation in the tumor suppressive effect of SCNN1B in GC.

Discussion

In this study, we identified that SCNN1B is readily expressed in normal gastric tissues, but is frequently silenced in GC cell lines and primary GC. Silencing of SCNN1B is associated with promoter methylation. Demethylation treatment with 5-Aza restored expression of SCNN1B, confirming that promoter hypermethylation mediates the transcriptional silencing of SCNN1B in GC.

SCNN1B gene silencing in GC suggests that SCNN1B may possess tumor suppressive function and its down-regulation may contribute to the development and progression of GC. Consistent with our hypothesis, the ectopic expression of SCNN1B in four GC cell lines (AGS, BGC823, MGC803 and MKN45) significantly suppressed cell proliferation in vitro; whilst its knockdown in MKN1 and NCI-N87 cells, which express endogenous SCNN1B, promoted cell viability. Tumor suppressive effect of SCNN1B was validated in vivo, as evidenced by the diminished growth of SCNN1B-expressing MKN45 and BGC823 cells in nude mice. SCNN1B suppressed GC cell proliferation through apoptosis induction and inhibition of cell cycle progression. SCNN1B overexpression induced apoptosis in GC cells by activating 17

both intrinsic and extrinsic apoptosis pathways, leading to the cleavage of caspase-8, caspase-9 and that of the downstream effectors, caspase-7 and PARP. Moreover,

SCNN1B inhibited cell cycle progression at G0/G1 phase, which was associated with up-regulation of p27kip1 and suppression of Cyclin D1 and CDK2. Cyclin D1/CDK2

kip1 forms an active complex that promotes G1-S transition; while p27 binds to and inhibits Cyclin D1/CDK2 activity (18). SCNN1B hence tips the balance of gene expression towards that of cell cycle arrest. Inhibition of cell growth in vivo by SCNN1B was confirmed by the reduced Ki-67 index in SCNN1B-expressing MKN45 xenografts. Metastasis is a major cause of cancer-related deaths. Here, we revealed that SCNN1B functions as a metastasis suppressor in GC by inhibiting cell migration and invasion, and concomitantly promoting cell adhesion. Taken together, these results indicate that SCNN1B functions as a tumor suppressor by inhibiting cell growth and metastasis in GC.

To further elucidate the mechanism of action of SCNN1B, we performed co-IP and mass spectrometry, which led to the identification of GRP78 as an interacting partner. The direct interaction between SCNN1B and GRP78 was validated by co-IP in GC cells and their co-localization by confocal immunofluorescence microscopy. Moreover, ectopic SCNN1B expression reduced protein expression of GRP78 without altering its mRNA expression. Instead, we showed that SCNN1B expression induced polyubiquitination of GRP78, which tagged the protein for degradation in the proteasome system (19). Re-expression of GRP78 in SCNN1B-expressing GC cells abrogated the inhibitory effects of SCNN1B on cell proliferation and cell migration; whereas GRP78 knockdown further aggravated SCNN1B-mediated growth inhibition. These data indicated that SCNN1B mediates its tumor suppressive effect by regulating GRP78, which is a master regulator of the UPR response. GRP78 is frequently up-regulated during cancer progression to counter UPR, maintain ER homeostasis and promote cell survival (15, 16). Taken together, interplay between SCNN1B and GRP78 regulates the stability of GRP78, which has serious repercussion on cell survival and migration/invasion. 18

GRP78 controls the UPR via the sequestration of IRE1α, PERK and ATF6 (20, 21). Whilst UPR is initiated as a pro-survival mechanism, sustained activation of this pathway induces apoptotic cell death and cell cycle arrest (22). We demonstrated that the ectopic expression of SCNN1B, through suppressing GRP78 expression, triggered the pro-apoptotic arm of the UPR response. This was exemplified by the increased PERK expression, which in turn, induced expression and nuclear localization of transcription factors ATF4 and CHOP in SCNN1B-expressing GC cells. Tunicamycin, an UPR inducer, exacerbated the inhibitory effect of SCNN1B on GC cell proliferation, implying that modulation of UPR response plays an important role in the tumor suppressive effect of SCNN1B. ATF4 and CHOP have been shown to induce apoptosis following prolonged stress, in part by increasing protein load and ATP depletion (23). CHOP also initiates expression of pro-apoptotic genes such as DR5 (24), BIM (25) and PUMA (26). UPR induction has also been associated with

G1-S cell cycle arrest via down-regulation of Cyclin D1 (27) and up-regulation of p27kip1 (28). GRP78 is also known to promote cancer metastasis, independent of UPR signalling (17). Collectively, our findings suggested that SCNN1B exerts a tumor suppressive effect through its involvement in regulating the expression of GRP78 and the UPR response signalling pathway.

Finally, we investigated the clinical importance of SCNN1B expression and promoter methylation in primary GC. Using a TMA cohort with 245 primary GC patients, we found that expression of SCNN1B was an independent prognostic factor of favorable patient survival by multivariate Cox regression analysis. Moreover, SCNN1B protein expression was significantly associated with survival benefit in late stage (TNM stages III/IV) GC. This was further validated by the association of high SCNN1B mRNA expression with improved survival of late stage GC patients in another two GC cohort (29,30). Gastric cancer varies greatly in clinical outcome depending on the aggressiveness of individual tumors. At present, TNM staging is the clinically most important predictor of patient survival in GC, and additional prognostic markers are 19

necessary to provide a more accurate assessment of disease outcomes. SCNN1B was identified to be associated with patient outcome, and therefore our data suggested that SCNN1B may serve as a novel prognostic marker for GC patients. Moreover, SCNN1B was found to suppress GC cell growth therefore it may serve as a therapeutic target of GC.

In summary, we identified for the first time that SCNN1B acts as a tumor suppressor through induction of apoptosis and cell cycle arrest, and inhibition of cell migration and invasion. We also uncovered that SCNN1B exerts its effect by direct interaction with GRP78, which led to its degradation and subsequent induction of UPR (Figure 7D). The expression status of SCNN1B may serve as prognostic markers in primary GC.

Acknowledgement: Dr. Zhinong Jiang, Sir Run Run Shaw Hospital, Hangzhou, China and Dr. Ye Cheng, Zhejiang Cancer Hospital, Hangzhou, China. These two pathologists both read and scored the TMA slides of GC patients.

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Figure legends

Figure 1. SCNN1B is silenced by promoter methylation in human GC. (A) Infinium Human Methylation 450K analysis revealed that CpGs within the SCNN1B locus are hypermethylated in GC cell lines as compared to a normal gastric epithelial cell line GES1 and normal gastric tissues. (B) CpGs at the SCNN1B promoter (-443 to -32 bp) were significantly methylated in GC. (C) SCNN1B mRNA was silenced in 13 out of 16 human GC cell lines, and its down-regulation was associated with promoter methylation as determined by methylation specific PCR (MSP). (D) Bisulfite-sequencing (BGS) was performed on the SCNN1B promoter and first exon CpG island. Dense methylation was observed in GC cell lines, but not in normal gastric tissues. (E) mRNA expression of SCNN1B was restored in GC cells after treatment with demethylation agent 5-Aza-2’-deoxycytidine (5-Aza) (left panel). SCNN1B mRNA expression was restored in the MKN45 cell line using 5-Aza plus trichostatin A (TSA) (right panel).

Figure 2. Promoter hypermethylation of SCNN1B leads to its down-regulation in GC tissues and SCNN1B expression serves as an independent predictor of GC-specific survival. (A) Expression of SCNN1B in both mRNA and protein level was significantly down-regulated in GC tumour tissues compared with paired adjacent normal gastric tissues. Its down-regulation was associated with promoter methylation as determined by methylation specific PCR (MSP). (B) Expression of SCNN1B mRNA in paired primary GC tissues in the Hong Kong (n=74, P<0.001) and the TCGA (n=34, P<0.001) cohort (left panel). Representative images of IHC staining of SCNN1B protein expression in GC and their adjacent normal tissues; Quantification of SCNN1B protein expression by scoring IHC staining in GC tissues (n=10, P<0.001) (right panel). (C) Representative the methylation status of SCNN1B in GC and adjacent normal tissues, which were confirmed by bisulfite genomic sequencing (BGS) (n=20). (D) TCGA dataset revealed an inverse correlation between SCNN1B mRNA expression and promoter methylation in primary GC. (E) Representative Kaplan– 24

Meier plots of the association between SCNN1B protein expression and disease-specific survival in GC. Intermediate or high SCNN1B expression had significantly longer survival (n=245, P<0.001). (F) Further stratification revealed that intermediate or high expression of SCNN1B predicted favorable survival in late stage (stage III/IV) GC (n=162, P=0.011) (right panel). But SCNN1B expression did not associate with disease-specific survival in early stage (stage I/II) GC (left panel).

Figure 3. SCNN1B inhibits GC cell growth and induced apoptosis. (A) Ectopic expression of SCNN1B in AGS, BGC823, MGC803 and MKN45 cell lines was confirmed by RT-PCR and Western blot. (B) SCNN1B overexpression inhibited colony formation and (C) cell proliferation in AGS, BGC823, MGC803 and MKN45 cells. (D) SCNN1B promoted the induction of apoptosis in GC cell lines, as shown by the Annexin V-PE/7-AAD assay (left panel) and the increased protein expression of the cleaved forms of caspase-8, caspase-9, caspase-7 and PARP (right panel). (E)

SCNN1B inhibited cell cycle progression at G0/G1 phase (left panel), and it increased the levels of p27Kip1 and p53 while reducing the expression of CDK2 and cyclin D1 (right panel). (F) Knockdown of SCNN1B in MKN1 cells was confirmed by RT-PCR and Western blot (left panel). SCNN1B knockdown increased colony formation (middle panel) and promoted cell cycle progression (right panel) in MKN1 cells.

Figure 4. SCNN1B regulates GC cell migration, invasion and adhesion. (A) Representative images of wound-healing assay indicated expression of SCNN1B suppressed cell migration in GC cell lines (AGS, BGC823, MGC803 and MKN45). (B) Representative images of cell adhesion assay showed expression of SCNN1B promoted GC cell adhesion. (C) Representative images of Matrigel invasion assay revealed ectopic expression of SCNN1B suppressed GC cell invasion. (D) siRNA mediated knockdown of SCNN1B in MKN1 cells enhanced wound closure. (E) siRNA mediated knockdown of SCNN1B in MKN1 cells decreased cell adhesion.

Figure 5. SCNN1B inhibits tumorigenicity in vivo. (A) Representative images of nude mice tumorigenicity assay with MKN45 cell line stably transfected with SCNN1B or empty vector. SCNN1B expression in the xenografts of MKN45-SCNN1B were confirmed by RT-PCR and Western blot. Tumor growth was slower and tumor weight was lower in mice injected with MKN45-SCNN1B cells than those with MKN45-emtpy vector cells. Ki-67 staining revealed a significant reduction in cell proliferation in MKN45 xenografts expressing SCNN1B by counting the proportion of Ki-67-positive cells. (B) Representative images of tumorigenicity assay with BGC823 cell line stably transfected with SCNN1B or empty vector in vivo. SCNN1B expression in the xenografts of BGC823-SCNN1B were confirmed by Western blot. Tumor growth was slower and tumor weight was lower in BGC823-SCNN1B group than BGC823-emtpy vector group.

Figure 6. SCNN1B interacts with GRP78 and mediates GRP78 protein degradation via polyubiquitination. (A) Immunoprecipitatant of Flag-tagged SCNN1B in HEK293 cells was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and followed by mass spectrometry (proteins of interest were indicated by arrow). Interaction between SCNN1B and GRP78 was confirmed by co-immunoprecipitation (co-IP) in AGS and BGC823 cells. (B) Representative images under confocal microscopy showed SCNN1B was located in the membrane and cytoplasm; whereas GRP78 was broadly expressed in membrane, cytoplasm and nucleus. Co-localization of SCNN1B and GRP78 was observed mainly in membrane and cytoplasm. (Green, GFP-tagged SCNN1B; Red, Myc-tagged GRP78; Blue, DAPI stained nuclei). (C) SCNN1B attenuated expression of GRP78 at protein level in GC cell line (left panel). Moreover, SCNN1B decreased the expression of GRP78 in the cytoplasm and membrane decided by Western blot (right panel). (D) MG132 (proteasome inhibitor), but not chloroquine (lysosome inhibitor), restored GRP78 protein levels in SCNN1B-overexpressing AGS cells (left panel), implying that the ubiquitin-proteasome pathway was involved in the degradation of GRP78. SCNN1B increased ubiquitin-mediated degradation of GRP78 (right panel). (E) SCNN1B 26

increased expression of PERK, XBP1s, ATF4, and CHOP. (F) SCNN1B sensitized GC cells to the cytotoxic effect of UPR inducer tunicamycin.

Figure 7. Tumor suppressive effect of SCNN1B is dependent on down-regulation of GRP78 (A) Co-transfection with empty vector or GRP78 in the control and SCNN1B expressing AGS cells revealed ectopic expression of GRP78 restored GRP78 protein levels in SCNN1B-overexpressing cells (left panel). Colony formation assay showed ectopic GRP78 expression restored the number of cell colonies in SCNN1B-expressing AGS cells (Right panel). (B) Ectopic GRP78 expression promoted wound closure in AGS-SCNN1B cells (P<0.01). (C) Knockdown of GRP78 in the control and SCNN1B over-expressing AGS cells was confirmed by Western blot (left panel). Colony formation assay showed siRNA-mediated knockdown of GRP78 together with ectopic SCNN1B expression inhibit cell growth of AGS cells (right panel). (D) Proposed mechanistic scheme of SCNN1B. SCNN1B directly interacts with GRP78 and promotes its ubiquitination-induced degradation. This leads to an UPR response involving induction of PERK, ATF4, CHOP, and XBP1s, which activates caspase-induced apoptosis; and suppression of cell migration and invasion. SCNN1B also induces p53/p27 and inhibited Cyclin D1/CDK2 expression, leading to cell growth arrest.

Sodium channel subunit SCNN1B suppresses gastric cancer growth and metastasis via GRP78 degradation

Jun Yu, Qian Yun, Chi Chun Wong, et al.

Cancer Res Published OnlineFirst February 15, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-1595

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