Sodium Channel Subunit SCNN1B Suppresses Gastric Cancer … · gastric cancer cell lines (AGS,...

Molecular and Cellular Pathobiology

Sodium Channel Subunit SCNN1B SuppressesGastric Cancer Growth and Metastasis viaGRP78 DegradationYun Qian1,2,3, Chi Chun Wong1, Jiaying Xu1, Huarong Chen1, Yanquan Zhang1,Wei Kang1,4, Hua Wang5, Li Zhang1,Weilin Li1, Eagle S.H. Chu1, Minnie Y.Y. Go1,Philip W.Y. Chiu6, Enders K.W. Ng6, Francis K.L. Chan1, Joseph J.Y. Sung1,Jianmin Si2,3, and Jun Yu1

Abstract

There remains a paucity of functional biomarkers in gastriccancer. Here, we report the identification of the sodium channelsubunit SCNN1B as a candidate biomarker in gastric cancer.SCNN1BmRNA expression was silenced commonly by promoterhypermethylation in gastric cancer cell lines and primary tumortissues. Tissuemicroarray analysis revealed that high expression ofSCNN1Bwas an independent prognostic factor for longer survivalin gastric cancer patients, especially those with late-stage disease.Functional studies demonstrated that SCNN1B overexpressionwas sufficient to suppress multiple features of cancer cell patho-physiology in vitro and in vivo. Mechanistic investigations revealedthat SCNN1B interacted with the endoplasmic reticulum chap-

erone, GRP78, and induced its degradation via polyubiquitina-tion, triggering the unfolded protein response (UPR) via activa-tion of PERK, ATF4, XBP1s, and C/EBP homologous protein andleading in turn to caspase-dependent apoptosis. Accordingly,SCNN1B sensitized gastric cancer cells to the UPR-inducing drugtunicamycin. GRP78 overexpression abolished the inhibitoryeffect of SCNN1B on cell growth and migration, whereas GRP78silencing aggravated growth inhibition by SCNN1B. In summary,our results identify SCNN1B as a tumor-suppressive function thattriggers UPR in gastric cancer cells, with implications for itspotential clinical applications as a survival biomarker in gastriccancer patients. Cancer Res; 77(8); 1968–82. �2017 AACR.

IntroductionGastric cancer is one of the most common human cancers.

Despite improvements in the surveillance and treatment ofgastric cancer, it remains a devastating disease with poor prog-nosis (1). Epigenetic dysregulation plays an important role ingastric carcinogenesis. Previous studies have shown that theinactivation of tumor suppressor genes by promoter DNAmethylation contributes to the pathogenesis of human gastric

cancer (2–8). To unveil novel tumor suppressor genes that aresilenced by epigenetic mechanisms in gastric cancer, we usedgenome-wide methylation array (Infinium Human Methylation450 K) to comprehensively profile CpG site methylation in fivegastric cancer cell lines (AGS, HGC27, MGC803, MKN1, andMKN45), an immortalized human gastric epithelial cell GES1and two normal gastric tissue samples. Using this approach, weidentified SCNN1B as a novel gene that is highly methylated inhuman gastric cancer, whose potential role in gastric cancerdevelopment is largely unknown.

SCNN1B is located on chromosome 16p12.2 and it encodesb-subunit of the epithelial sodium channel (ENaC). SCNN1B isa part of a multiprotein complex consisting of three subunits(a, b, and g) that controls fluid and electrolyte transport acrossepithelia in diverse organs. SCNN1B is classified as a membranechannel, but accumulating evidence also indicates that ENaCsubunits, including SCNN1B, participate in cellular differenti-ation (9–11). SCNN1A, which encodes the a-subunit of ENaC,has been shown to be silenced by promoter methylation inneuroblastoma and breast cancer (12, 13). However, the func-tional importance of SCNN1B in human cancer remains unex-plored. In this study, we identified frequent silencing ofSCNN1B in human gastric cancer, which was associated withpromoter methylation. We demonstrated a significant correla-tion between the silence of SCNN1B protein expression andpoor disease-specific survival of gastric cancer patients. Werevealed that SCNN1B suppresses gastric cancer growth byinducing apoptosis and cell-cycle arrest and inhibiting meta-stasis abilities. The tumor-suppressive effect of SCNN1B was

1Institute of Digestive Disease and Department of Medicine and Therapeutics,State Key Laboratory of Digestive Disease, Li Ka Shing Institute of HealthSciences, CUHK Shenzhen Research Institute, The Chinese University of HongKong, Hong Kong, China. 2Department of Gastroenterology, Sir Run Run ShawHospital, School of Medicine, Zhejiang University, Hangzhou, China. 3Institute ofGastroenterology, Zhejiang University, Hangzhou, China. 4Department of Ana-tomical and Cellular Pathology, The Chinese University of Hong Kong, HongKong, China. 5School of Biomedical Science, The Chinese University of HongKong, Hong Kong, China. 6Department of Surgery, The Chinese University ofHong Kong, Hong Kong, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Y. Qian and C.C. Wong contributed equally to this article.

Corresponding Author: Jun Yu, The Chinese University of Hong Kong, Rm707A,Li Ka ShingMedical Sciences Building, Prince ofWales Hospital, Shatin, NT, HongKong, China. Phone: 852-3763-6099; Fax: 852-2144-5330; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-1595

�2017 American Association for Cancer Research.

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found to be mediated via (i) the direct interaction with GRP78,a chaperone with oncogenic properties; (ii) the reduction ofGRP78 protein by inducing its polyubiquitination and protea-some-mediated degradation; and (iii) the induction of theunfolded protein response (UPR) response, which activatesPERK, ATF4, XBP1s, and C/EBP homologous protein (CHOP),leading to caspase-dependent apoptosis and cell-cycle arrest.Moreover, tissue microarray (TMA) analysis of 245 gastriccancer patients revealed that high SCNN1B expression is anindependent prognostic factor that predicts better survival ofgastric cancer patients.

Materials and MethodsCell culture

Sixteen gastric cancer cell lines and a normal gastric epithe-lial cell line were used in this study: AGS, KATOIII, MKN45,and NCI-N87 cells were obtained from the ATCC; MKN1,MKN74, SNU1, SNU638, SNU719, and YCC10 cells wereobtained from the Korean Cell Line Bank; MKN7 cells wereobtained from RIKEN Cell Bank; BGC823, HGC27, MGC803,SGC7901, and normal gastric epithelial cell line GES1 wereobtained from the Cell Bank of Chinese Academy of Sciences(Shanghai, China). All cells were purchased between 2014 and2015, and routinely cultured in DMEM containing 10% FBSand penicillin–streptomycin.

SCNN1B ectopic expression and knockdownThe full-length ORF of SCNN1B was cloned into pcDNA3.1,

pCMV4-FLAG, and pEGFP-N1 vectors. Transfection was per-formed with Lipofectamine 2000 (Life Technologies). Cell linesstably expressing SCNN1B were obtained after selection withneomycin (G418, Life Technologies) for at least 2weeks. SCNN1BsiRNA were purchased from RiboBio Co. Ltd and transfected intoMKN1 and NCI-N87 cells using Lipofectamine 2000.

Colony formation and cell growth curve assaysCells were plated in 6-well plates at 1,000 cells per well

in complete DMEM. Medium was changed every 3 to 4 days.At the endpoint, cells were stained with 0.1% Crystal violetand the number of colonies consisting of >50 cells werecounted. Cell growth curve was performed using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay (Sigma-Aldrich).

Apoptosis and cell-cycle analysisCells were plated in 12-well plates and serum-starved over-

night. Annexin V-PE/7-aminoactinomycin D (7-AAD) staining kit(BD Biosciences) was used to determine cell apoptosis. For cellcycle, cells were serum-starved for 24 hours and then stimulatedwith complete medium for 4 to 12 hours. Cell-cycle distributionwas then assessedbyflowcytometry after stainingwithpropidiumiodide (Life Technologies).

Wound-healing assayConfluent cultures in 6-well plateswere scratchedwith sterile P-

200 pipette tips, washed, and cultured in DMEM containing 2%FBS. Cells were photographed after 0, 12, 24, and 48 hours,respectively. Wound closure (%) was evaluated by the TScratchsoftware.

Invasion assayCell invasion was determined using BD BioCoat Matrigel

Invasion Chamber (BD Biosciences). Cells (5 � 104/well) wereseeded onto the top chamber in serum-free DMEM. CompleteDMEM (supplemented with 10% FBS) was added to the bottomchamber as a chemoattractant. After 48 hours, cells that hadinvaded through the membrane were stained with 0.1% Crystalviolet and counted.

Adhesion assayCells (0.5–1� 105/well) were seeded onto 96-well plates. After

30 and 60minutes, themediumwas aspirated, then the cells werewashedwith PBS and stainedwith 0.1%Crystal violet. TheCrystalviolet was dissolved in 10% acetic acid overnight and absorbancewas measured at 540 nm.

ImmunofluorescenceCells were seeded onto coverslips in a 6-well plate and trans-

fectedwith GFP-tagged SCNN1B andMyc-taggedGRP78. Twenty-four hours after transfection, cells were fixed with 4% parafor-maldehyde and permeabilized with 0.2% Triton X-100, blockedin 5%BSA in PBS, and incubatedwith anti-Myc (1:4,000dilution)overnight at 4�C, followed by anti-mouse IgG secondary antibodyconjugated with Alexa Fluor 594 (1:400 dilution, Yeasen) in thedark for 1 hour. Cells were then mounted with ProLong GoldAntifade Mountant with DAPI (Life Technologies). Images werecaptured using a Carl Zeiss LSM 780 confocal laser scanningmicroscope (Carl Zeiss AG).

Human samplesPaired primary gastric tumors and adjacent normal gastric

tissues were collected immediately after surgical resection at thePrince of Wales Hospital (Hong Kong, China). The specimenswere snap-frozen in liquid nitrogen and stored at�80�Candwerealso fixed in 10% formalin and embedded in paraffin for routinehistologic examination. Biopsies from 3 cases of normal mucosaobtained during gastroscopy were recruited as healthy controls,which were confirmed by an experienced pathologist at the Princeof Wales Hospital. All patients gave informed consent, and thestudy protocol was approved by the Clinical Research EthicsCommittee of the Chinese University of Hong Kong (Hong Kong,China).

TMA assayTMA was generated from formalin-fixed, paraffin-embedded

archived tissue samples of 245 patients with gastric cancer priorto radiotherapy/chemotherapy, which were collected at thePrince of Wales Hospital (Hong Kong, China; ref. 14), witha median follow-up time of 40.8 months. All subjects provid-ed informed consent for obtaining the specimens. TMA wasstained with a commercially available anti-SCNN1B anti-body (HPA015612, Sigma-Aldrich). Anti-SCNN1B antibody(HPA015612) was confirmed by antibody specificity analysiswith protein arrays, with single peak corresponding to inter-action only with its own antigen. Cytoplasmic expression ofSCNN1B was assessed by H-score. The proportion score was inthe light of proportion of cancer cells with positive cytoplasmicstaining (0, no positive staining; 1, in 10% or fewer cells; 2, inbetween 10% and 25% cells; 3, in between 25% and 50% cells;4, in more than 50% cells). The intensity score was assignedfor the average intensity of cancer cells with positive staining

SCNN1B as a Novel Tumor Suppressor

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(0, none; 1, weak; 2, intermediate; 3, strong). The IHC score ofSCNN1B was calculated by the following formula: IHC score ¼proportional score (0–4) � intensity score (0–3), ranging from0 to 12. Finally, the cytoplasmic expression of SCNN1B ingastric cancer tissue was divided into 3 groups according to IHCscore (low, �3; intermediate, 4–6; high, 7–12). The resultswere scored independently by two pathologists and the averageof the two values was taken.

Coimmunoprecipitation–mass spectrometryCells transiently transfected with SCNN1B-FLAG or empty

vector were lysed with ice-cold RIPA lysis buffer (50 mmol/LTris-Cl, 150 mmol/L NaCl, 1% NP-40, 0.5% sodium deoxycho-late, and 1% SDS, pH 8.0) supplemented with protease inhibitors(Roche). Total proteins were immunoprecipitated using 2 mg ofanti-Flag (F1804, Sigma-Aldrich) and bound to 40 mL of ProteinG-Agarose (Santa Cruz Biotechnology). After washing 5 timeswith RIPA buffer, bound proteins were elutedwith loading buffer,separated by SDS-PAGE, and visualized by silver staining. Proteinbands of interest in-gel were digested, and subjected to LC/MS-MS(ABI4800 MALDI TOF/TOF, Applied Biosystems). The MS frag-ment spectra were analyzed using Mascot software (Matrix Sci-ence). To confirm the interaction of SCNN1B with GRP78,immune complexes were precipitated by anti-Flag and analyzedby Western blot analysis using anti-Flag and anti-GRP78(Sc-13968, Santa Cruz Biotechnology).

Ubiquitination assayAGS cells stably transfected with SCNN1B expression vector or

empty vector were lysed with RIPA buffer supplemented withprotease inhibitors. Immunoprecipitation was performed usinganti-GRP78 or control IgG, respectively. Immunoprecipitatedproteins were analyzed by Western blot analysis using anti-ubi-quitin (3936, Cell Signaling Technology).

Subcutaneous xenograft modelBGC823 (1� 107 cells/0.1mLPBS) andMKN45 (1� 106 cells/

0.1 mL PBS) cells stably expressing the control vector or SCNN1Bwere injected subcutaneously into the left and right dorsal flank of4- to 6-week-old female Balb/c nude mice (n ¼ 6/group), respec-tively. Tumor size was measured every 2 days for 2–3 weeks usinga digital caliper. Tumor volume (V) was estimated by measuringthe longest diameter (L) and shortest diameter (W) of the tumorand calculated by formula V ¼ 0.5 � L � W2. At the endpoint,tumorswere harvested andweighted. All experimental procedureswere approved by the Animal Ethics Committee of the ChineseUniversity of Hong Kong (Hong Kong, China).

Statistical analysisAll the results were expressed as mean � SEM (continuous

variables) or described as frequency and percentage (categoricaldata). To compare the difference between two groups, indepen-dent sample t test or Mann–Whitney U test was used. Thedifference between growth rates was determined by ANOVA withrepeated-measures ANOVA. The Pearson c2test or Fisher exact testwas used for analysis of the associations between patient clini-copathologic characteristics and SCNN1B expression. Kaplan–Meier analysis and log-rank test were performed to evaluate theassociation between SCNN1B expression and disease-specificsurvival. Cox proportional hazards regression model was per-formed to assess the prognostic value of SCNN1B expression. All

the statistical analyses were performed using GraphPad Prism,version 6.0 (GraphPad Software) or SPSS, version 20.0 (SPSSInc.). P < 0.05 was considered statistically significant.

ResultsGenome-wide methylation analysis identified SCNN1Bpromoter is densely methylated in human gastric cancer

Using the Infinium Human Methylation 450 K array, weinterrogated genome-wideCpGmethylation infivehumangastriccancer cell lines (AGS, HGC27,MGC803,MKN1, andMKN45) ascompared with normal gastric epithelial cell line GES1 andnormal gastric tissues (Fig. 1A). Using stringent criteria, weidentified SCNN1B to be preferentiallymethylated at its promoterin gastric cancer (Fig. 1B).

SCNN1B is silenced in gastric cancer cell lines and primarygastric cancer by promoter methylation

We initially examined SCNN1B mRNA expression in humannormal tissues, and found that SCNN1B was widely expressed inmost human normal tissues with strong expression in the stom-ach (Supplementary Fig. S1). On the other hand, SCNN1BmRNAexpression was silenced in 13 of 16 (81.3%) gastric cancer celllines (Fig. 1C); only MKN1, MKN7, and NCI-N87 cells linesexpressed significant levels of SCNN1B mRNA (Fig. 1C; Supple-mentary Fig. S2). To determine the role of promoter methylationin silencing of SCNN1B, we evaluated its promoter methylationby methylation-specific PCR (MSP) and bisulfite genomicsequencing (BGS). MSP analysis revealed dense SCNN1B pro-moter methylation in all gastric cancer cell lines with silencedSCNN1B expression (Fig. 1C). BGS analysis of 38 CpG sites inSCNN1B promoter and the first exon showed dense methylation(average methylation > 50%) in the SCNN1B-silenced gastriccancer cell lines examined, but not in SCNN1B-expressing MKN1cells and normal gastric tissues (Fig. 1D; Supplementary Fig. S3).To test whether promoter methylation directly mediates thesilencing of SCNN1B, six gastric cancer cell lines with silencedSCNN1B expression were treated with DNA methyltransferaseinhibitor, 5-Aza-20-deoxycytidine (5-Aza). 5-Aza restoredSCNN1B expression in all six cell lines, indicating that promotermethylation contributes to the transcriptional silencing ofSCNN1B (Fig. 1E). In addition, treatment with 5-Aza plus histonedeacetylase inhibitor trichostatin A (TSA) could fully restoreSCNN1B expression in MKN45 cells with moderate promotermethylation (Fig. 1E).

We evaluated mRNA and protein expression of SCNN1B ingastric tissues from 74 primary gastric cancer patients. SCNN1BmRNA was significantly downregulated in gastric cancer as com-pared with paired adjacent normal gastric tissues (P < 0.0001;Fig. 2A and B). SCNN1B mRNA expression was also downregu-lated in gastric cancer in the The Cancer Genome Atlas (TCGA)cohort (n ¼ 34; P < 0.0001; Fig. 2B). Western blot analysis andIHC confirmed the reduced expression of SCNN1B in gastriccancer as compared with adjacent normal tissues (n ¼ 10;P < 0.001; Fig. 2A and B). We next examined the methylationstatus of SCNN1B in primary gastric cancer. MSP and BGSanalysis demonstrated that SCNN1B promoter methylation wassignificantly higher in gastric cancer as compared with adjacentnormal tissues (Fig. 2A and C). None of the normal gastricbiopsies showed SCNN1B promoter methylation. These dataimplied that SCNN1B is silenced by promoter methylation in

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Figure 1.

SCNN1B is silenced by promoter methylation in human gastric cancer. A, Infinium Human Methylation 450K analysis revealed that CpGs within theSCNN1B locus are hypermethylated in gastric cancer cell lines as compared with a normal gastric epithelial cell line GES1 and normal gastric tissues. B, CpGsat the SCNN1B promoter (�443 to �32 bp) were significantly methylated in gastric cancer. C, SCNN1B mRNA was silenced in 13 of 16 human gastriccancer cell lines, and its downregulation was associated with promoter methylation as determined by MSP. D, BGS was performed on the SCNN1Bpromoter and first exon CpG island. Dense methylation was observed in gastric cancer cell lines, but not in normal gastric tissues. E, mRNA expressionof SCNN1B was restored in gastric cancer cells after treatment with demethylating agent 5-Aza (left). SCNN1B mRNA expression was restored in theMKN45 cell line using 5-Aza plus TSA (right). TSS, transcription start site.






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gastric cancer. Consistent with our data, analysis of the TCGAdataset revealed an inverse correlation between SCNN1BmRNA expression and promoter methylation in gastric cancer(P < 0.001; Fig. 2D).

SCNN1B expression is an independent predictor of favorableoutcome in gastric cancer patients

To evaluate the association of SCNN1B expression with clin-icopathologic features and clinical outcomes, we assessed theSCNN1B protein expression in gastric cancer utilizing a gastriccancer TMA (n¼ 245). SCNN1B cytoplasmic expression showed asignificant correlation with TNM stage (P < 0.001) and lymphaticmetastasis (P ¼ 0.036), but had no correlation with age, gender,H. pylori infection, histologic Lauren classification, or tumor grade(Supplementary Table S1). In univariate Cox regression analysis,an intermediate or high cytoplasmic SCNN1B score was associ-atedwith better disease-specific survival [intermediate: HR, 0.482;95% confidence interval (CI), 0.320–0.726, P < 0.001; high: HR,0.247, 95% CI, 0.091–0.674, P ¼ 0.006]. Apart from SCNN1Bexpression, age (P ¼ 0.048), histologic Lauren classification(P < 0.001), tumor grade (P ¼ 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, histologic Lauren classification, tumor grade, and TNMstage, SCNN1B expression was found to be an independentprognostic factor for disease-specific survival [intermediate: HR,0.547; 95%confidence interval (CI), 0.360–0.829; P¼ 0.005; andhigh:HR, 0.353; 95%CI, 0.128–0.971;P¼0.044] bymultivariateCox proportional hazards regression analysis (SupplementaryTable S2). As shown by Kaplan–Meier curves, gastric cancerpatients with intermediate or high SCNN1B protein expressionhad significantly longer survival (P < 0.001; Fig. 2E). Furtherstratification of the TMA cohort into early stage (TNM stage I/II)and late stage (TNM stage III/IV) revealed that intermediate orhigh protein expression of SCNN1B was associated with bettersurvival in late-stage gastric cancer (P¼ 0.011; Fig. 2F). Analysis ofanother two independent gastric cancer cohorts (GSE62254 andGSE14210) also showed that high SCNN1B mRNA expressionwas associated with better survival in late-stage gastric cancer(Supplementary Fig. S4; Supplementary Tables S3 and S4). Theseresults indicate that high SCNN1B expression predicts a favorableprognosis in patients with gastric cancer.

SCNN1B suppresses gastric cancer cell growth through theinduction of apoptosis and cell-cycle arrest

The frequent silencing of SCNN1B in gastric cancer andits association with patient survival prompted us to hypothe-

size that SCNN1B functions as a tumor suppressor. To thisend, we generated four gastric cancer cell lines (AGS, BGC823,MGC803, and MKN45) with stable SCNN1B expression.Ectopic expression of SCNN1B was validated by RT-PCR andWestern blot analysis (Fig. 3A), which was comparable withthat of normal gastric tissues (Supplementary Fig. S5).SCNN1B overexpression suppressed colony formation abilityby 55%–80% as compared with empty vector–transfectedcells in all four gastric cancer cell lines (Fig. 3B; P < 0.01).Consistently, cell growth curve assay revealed that ectopicSCNN1B expression inhibited viability in these cell lines(Fig. 3C, P < 0.001).

To determine the cytokinetic effect of SCNN1B on gastriccancer cells, we analyzed apoptosis and cell-cycle distributionby flow cytometry. Overexpression of SCNN1B led to a signif-icant 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 dualstaining (Fig. 3D). Induction of apoptosis by SCNN1B wasconfirmed by the elevated expression of key apoptosis markerssuch as cleaved forms of caspase-9, caspase-8, caspase-7, andPARP, as determined by Western blot analysis (Fig. 3D). Wealso observed an increased accumulation of gastric cancer cellsin the G1 phase (P < 0.05) and a reduction of S-phase popu-lation (P < 0.05) following ectopic SCNN1B expression (Fig.3E). Consistent with G1 arrest, we found that SCNN1Bincreased the expression of G1-phase gatekeepers, p27Kip1 andp53, while reducing expression of cyclin D1 and CDK2, both ofwhich are important for G1 progression (Fig. 3E). Next, weperformed loss-of-function experiments using two indepen-dent SCNN1B-targeted siRNAs to knockdown endogenousSCNN1B in MKN1 and NCI-N87 cells (Fig. 3F; SupplementaryFig. S6A). The knockdown of SCNN1B increased colony for-mation ability (P < 0.001) and promoted cell-cycle progressionin MKN1 and NCI-N87 cells (P < 0.01; Fig. 3F; SupplementaryFig. S6B and S6C). These data indicate that SCNN1B suppressesgastric cancer cell proliferation.

SCNN1B regulates gastric cancer cell migration, invasion,and adhesion

In light of the association between SCNN1B expressionand metastasis of gastric cancer patients, we next ask whetherSCNN1B has an effect on cell migration, adhesion, and invasion.SCNN1B overexpression markedly suppressed cell migration inAGS, BGC823, MGC803, andMKN45 cell lines by wound-healingassay. Quantitative analysis demonstrated a significant impair-ment in wound closure at different time points (P < 0.001) in

Figure 2.Promoter hypermethylation of SCNN1B leads to its downregulation in gastric cancer tissues and SCNN1B expression serves as an independent predictorof gastric cancer–specific survival. A, Expression of SCNN1B in both mRNA and protein level was significantly downregulated in gastric cancer tumortissues (T) compared with paired adjacent normal gastric tissues (N). Its downregulation was associated with promoter methylation as determined byMSP. B, Expression of SCNN1B mRNA in paired primary gastric cancer tissues in the Hong Kong (n ¼ 74, P < 0.001) and the TCGA (n ¼ 34, P < 0.001)cohort (left). Representative images of IHC staining of SCNN1B protein expression in gastric cancer and their adjacent normal tissues; quantificationof SCNN1B protein expression by scoring IHC staining in gastric cancer tissues (n ¼ 10; P < 0.001; right). C, Representative methylation status ofSCNN1B in gastric cancer and adjacent normal tissues, which was confirmed by BGS (n ¼ 20). D, TCGA dataset revealed an inverse correlationbetween SCNN1B mRNA expression and promoter methylation in primary gastric cancer. E, Representative Kaplan–Meier plots of the associationbetween SCNN1B protein expression and disease-specific survival in gastric cancer. Intermediate or high SCNN1B expression had significantly longersurvival (n ¼ 245; P < 0.001). F, Further stratification revealed that intermediate or high expression of SCNN1B predicted favorable survival inlate-stage (stage III/IV) gastric cancer (n ¼ 162; P ¼ 0.011; right), but SCNN1B expression did not associate with disease-specific survival in early-stage(stage I/II) gastric cancer (left).






Figure 3.

SCNN1B inhibits gastric cancer cell-growth and induced apoptosis. A, Ectopic expression of SCNN1B in AGS, BGC823, MGC803, and MKN45 cell lineswas confirmed by RT-PCR and Western blot analysis. SCNN1B overexpression inhibited colony formation (B) and cell proliferation (C) in AGS, BGC823,MGC803, and MKN45 cells. D, SCNN1B promoted the induction of apoptosis in gastric cancer cell lines, as shown by the Annexin V-PE/7-AAD assay(left) and the increased protein expression of the cleaved forms of caspase-8, caspase-9, caspase-7, and PARP (right). E, SCNN1B inhibited cell-cycleprogression at G0–G1 phase (left), and it increased the levels of p27Kip1 and p53 while reducing the expression of CDK2 and cyclin D1 (right). F, Knockdownof SCNN1B in MKN1 cells was confirmed by RT-PCR and Western blot analysis (left). SCNN1B knockdown increased colony formation (middle) andpromoted cell-cycle progression (right) in MKN1 cells.

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Figure 4.

SCNN1B regulates gastric cancer cell migration, invasion, and adhesion. A, Representative images of wound-healing assay indicated expression ofSCNN1B suppressed cell migration in gastric cancer cell lines (AGS, BGC823, MGC803, and MKN45). B, Representative images of cell-adhesion assayshowed that expression of SCNN1B promoted gastric cancer cell adhesion. C, Representative images of Matrigel invasion assay revealed ectopic expressionof SCNN1B-suppressed gastric cancer cell invasion. D, siRNA-mediated knockdown of SCNN1B in MKN1 cells enhanced wound closure. E, siRNA-mediatedknockdown of SCNN1B in MKN1 cells decreased cell adhesion.






SCNN1B-overexpressing cells, thereby suggesting that SCNN1Bnegatively regulates cell migration (Fig. 4A). SCNN1B also pro-moted cell adhesion in all four gastric cancer cell lines (Fig. 4B). Inaddition, Matrigel invasion assay revealed that ectopic expression

of SCNN1B suppressed cell invasion in AGS, BGC823, andMGC803 cells by over 50% (P < 0.001; Fig. 4C). Conversely,siRNA-mediated SCNN1B silencing in MKN1 and NCI-N87 cellsresulted in enhanced wound closure (P < 0.001), but decreased

Figure 5.

SCNN1B inhibits tumorigenicity in vivo. A, Representative images of nude mice tumorigenicity assay with MKN45 cell line stably transfected with SCNN1B orempty vector. SCNN1B expression in the xenografts of MKN45-SCNN1B was confirmed by RT-PCR and Western blot analysis. Tumor growth wasslower and tumor weight was lower in mice injected with MKN45-SCNN1B cells than those with MKN45-emtpy vector cells. Ki-67 staining revealed a significantreduction in cell proliferation in MKN45 xenografts expressing SCNN1B by counting the proportion of Ki-67–positive cells. B, Representative images oftumorigenicity assay with BGC823 cell line stably transfected with SCNN1B or empty vector in vivo. SCNN1B expression in the xenografts of BGC823-SCNN1Bwas confirmed by Western blot analysis. Tumor growth was slower and tumor weight was lower in BGC823-SCNN1B group than BGC823-emtpy vector group.

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Figure 6.

SCNN1B interacts with GRP78 andmediates GRP78 protein degradation via polyubiquitination.A, Immunoprecipitation (IP) of Flag-tagged SCNN1B in HEK293 cellswas analyzedbySDS-PAGEand followedbymass spectrometry (proteins of interest are indicatedby arrow). Interaction between SCNN1B andGRP78was confirmedby co-IP in AGS and BGC823 cells. B, Representative images under confocal microscopy showed that SCNN1B was located in the membrane and cytoplasm,whereas GRP78 was broadly expressed in membrane (M), cytoplasm (C), and nucleus (N). Colocalization of SCNN1B and GRP78 was observed mainly in membraneand cytoplasm. Green, GFP-tagged SCNN1B; red, Myc-tagged GRP78; blue, DAPI-stained nuclei. C, SCNN1B attenuated expression of GRP78 at protein level ingastric cancer cell lines (left). Moreover, SCNN1B decreased the expression of GRP78 in the cytoplasm and membrane as demonstrated by Western blotanalysis (right).D,MG132 (proteasome inhibitor), but not chloroquine (lysosome inhibitor), restoredGRP78 protein levels in SCNN1B-overexpressing AGS cells (left),implying that the ubiquitin–proteasome pathway was involved in the degradation of GRP78. SCNN1B increased ubiquitin-mediated degradation of GRP78 (right).E, SCNN1B increased the expression of PERK, XBP1s, ATF4, and CHOP. F, SCNN1B sensitized gastric cancer cells to the cytotoxic effect of UPR inducer tunicamycin.






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cell adhesion (P < 0.05) as compared with control (Fig. 4D and E;Supplementary Fig. S6D and S6E). Thus, SCNN1B reduces themetastatic ability of gastric cancer cells by inhibiting cell migrationand invasion, while promoting cell adhesion.

Ectopic SCNN1B expression inhibits tumorigenicity in nudemice

In light of our in vitro results, we evaluated the impact ofectopic SCNN1B expression in the nude mice tumorigenicityassay. MKN45 and BGC823 cell lines with stable expression ofempty vector or SCNN1B were injected into the left and rightflanks of nude mice, respectively. As shown in Fig. 5, tumorgrowth was significantly slower in mice injected with MKN45-SCNN1B cells than those with MKN45-emtpy vector cells (P <0.01; Fig. 5A) and in mice with BGC823-SCNN1B cells, thanthose with BGC823-emtpy vector cells (P < 0.01; Fig. 5B). Theaverage tumor weight at sacrifice was significantly lower inMKN45-SCNN1B mice (P < 0.05; Fig. 5A) and in BGC823-SCNN1B mice (P < 0.05; Fig. 5B), as compared with theircorresponding control mice. Ectopic SCNN1B expression in thetumor xenografts of MKN45-SCNN1B and BGC823-SCNN1Bwere confirmed by RT-PCR and Western blot analysis (Fig. 5Aand B; Supplementary Fig. S7). Ki-67 staining revealed a sig-nificant reduction in cell proliferation in MKN45 tumors expres-sing SCNN1B (P < 0.05; Fig. 5A). These results supported atumor-suppressive role for SCNN1B.

SCNN1B interacts with GRP78 and mediates GRP78 proteindegradation via polyubiquitination

To further elucidate themolecular mechanism underlying thetumor-suppressive effect of SCNN1B, we performed coimmu-noprecipitation (co-IP) of Flag-tagged SCNN1B in HEK293cells, followed by mass spectrometry to identify its bindingpartners (Fig. 6A). The 78-kDa glucose-regulated protein(GRP78) was identified as a potential interacting partner forSCNN1B. GRP78 is a stress-inducible chaperone that normallyresides in endoplasmic reticulum (ER); however, recentadvances have shown that GRP78 plays an oncogenic role incancer via supporting cell proliferation, invasion, and metas-tasis, and inhibition of apoptosis (15–17). We validated theinteraction between SCNN1B and GRP78 in AGS and BGC823cells (Fig. 6A), in which GRP78 was coimmunoprecipitated byFlag-tagged SCNN1B in both cell lines. We next evaluatedcolocalization of SCNN1B and GRP78 by immunofluorescencein AGS, BGC823, and MKN45 cell lines (Fig. 6B). Confocalmicroscopy images showed that GFP-tagged SCNN1B wasfound in the membrane and cytoplasm; whereas Myc-taggedGRP78 was expressed in membrane and cytoplasm. Colocaliza-tion of SCNN1B and GRP78 was observed mainly in the

cytoplasm. These results indicate that SCNN1B is a bindingpartner of GRP78.

We next assessed the interplay between SCNN1B and GRP78.GRP78 mRNA levels were not altered following the ectopicexpression of SCNN1B (Supplementary Fig. S8) On the otherhand, SCNN1B expression strongly attenuated expression ofGRP78 at protein level in gastric cancer cell lines (Fig. 6C).Moreover, SCNN1B decreased the expression of GRP78 in thecytoplasm and membrane that was consistent with their coloca-lization, thus implying that SCNN1B might regulate GRP78 viaprotein degradation (Fig. 6C).

Protein degradation in eukaryotic cells is mediated by twomajor pathways: ubiquitin–proteasome and autophagy–lyso-somal pathways. To pinpoint the mechanism of SCNN1B-induced GRP78 degradation, we treated SCNN1B-overexpressingAGS cells with inhibitors of the proteasome (MG132) and lyso-some (chloroquine). MG132, but not chloroquine, restoredGRP78 protein levels in SCNN1B-overexpressing AGS cells(Fig. 6D), implying that the ubiquitin–proteasome pathwaywas involved in the degradation of GRP78. To validate this, weexamined polyubiquitination of GRP78 with or without ectopicSCNN1B expression (Fig. 6D). Indeed, SCNN1B increased poly-ubiquitination of GRP78. Collectively, these data indicatedthat SCNN1B directly interacts with GRP78 and mediates GRP78degradation via ubiquitination.

Downregulation of GRP78 by SCNN1B induces cell deathvia UPR

GRP78 is a central regulator of the UPR by sequestration ofthree canonical branches, PERK-eIF2a-ATF4, IRE1a-XBP1s,and ATF6 pathways. Given that GRP78 is downregulated bySCNN1B, we evaluated the activation of the three UPR signal-ing pathways. Increased expression of PERK, XBP1s, and ATF4were demonstrated in SCNN1B-overexpressing gastric cancercell lines by Western blot analysis (Fig. 6E). In addition,nuclear abundance of ATF4 and XBP1s was simultaneouslyinduced in SCNN1B-expressing gastric cancer cells, suggestingthat these transcription factors were activated (Fig. 6E). We alsoobserved the upregulation of CHOP, a key mediator of UPR-mediated apoptotic pathway, in SCNN1B-overexpressinggastric cancer cell lines (Fig. 6E). To test whether inductionof UPR plays an important role in tumor-suppressive effect ofSCNN1B, we coincubated control and SCNN1B-expressinggastric cancer cells with the UPR stress inducer tunicamycin.We found that overexpression of SCNN1B sensitized AGS andMKN45 cells to the cytotoxic effect of tunicamycin as com-pared with controls. IC50 values of tunicamycin in AGS-emptyvector and AGS-SCNN1B cells were 368 and 234 ng/mL,respectively. A similar trend was observed in the MKN45 cellline, where empty vector cells (IC50: 1,175 ng/mL) were less

Figure 7.Tumor-suppressive effect of SCNN1B is dependent on downregulation of GRP78. A, Cotransfection with empty vector or GRP78 in the control andSCNN1B-expressing AGS cells revealed ectopic expression of GRP78-restored GRP78 protein levels in SCNN1B-overexpressing cells (left). Colonyformation assay showed ectopic GRP78 expression restored the number of cell colonies in SCNN1B-expressing AGS cells (right). B, Ectopic GRP78expression promoted wound closure in AGS-SCNN1B cells (P < 0.01). C, Knockdown of GRP78 in the control and SCNN1B-overexpressing AGS cellswas confirmed by Western blot analysis (left). Colony formation assay showed siRNA-mediated knockdown of GRP78 together with ectopicSCNN1B expression inhibit cell growth of AGS cells (right). D, Proposed mechanistic scheme of SCNN1B. SCNN1B directly interacts with GRP78 andpromotes its ubiquitination-induced degradation. This leads to an UPR response involving induction of PERK, ATF4, CHOP, and XBP1s, whichactivates caspase-induced apoptosis, and suppression of cell migration and invasion. SCNN1B also induced p53/p27 and inhibited cyclin D1/CDK2expression, leading to cell growth arrest.






sensitive than SCNN1B-expressing cells (IC50: 886 ng/mL) totunicamycin (Fig. 6F). Collectively, induction of UPR plays animportant role in tumor-suppressive function of SCNN1B ingastric cancer.

The tumor-suppressive effect of SCNN1B is dependent ondownregulation of GRP78. Given that SCNN1B abrogatedGRP78 expression through polyubiquitination, we next con-ducted rescue experiments in which we cotransfected the con-trol and SCNN1B-expressing gastric cancer cells with emptyvector or GRP78. We first evaluated the effect of ectopic GRP78expression on cell proliferation. As shown in Fig. 7A, ectopicexpression of GRP78 restored GRP78 protein levels inSCNN1B-overexpressing cells. Moreover, colony formationassay showed that GRP78 restored the number of cell coloniesin SCNN1B-expressing cells to baseline levels in AGS cells (P <0.01), whereas GRP78 overexpression did not promote colonyformation in control cells (Fig. 7A). This indicated that growth-suppressive effect of SCNN1B is mediated by downregulationof GRP78. We next investigated the effect of GRP78 overexpres-sion on metastatic capacity of SCNN1B-overexpressing AGScells. While control AGS cells had comparable wound closurerate irrespective of GRP78 expression, GRP78 promoted woundclosure in SCNN1B-overexpressing AGS cells (P < 0.001;Fig. 7B), implying that SCNN1B-mediated degradation ofGRP78 contributed to its antimetastatic effect in gastric cancercells. In contrast, siRNA-mediated knockdown of GRP78 wasadditive with ectopic SCNN1B expression to suppress GRP78expression and inhibit AGS cell growth as compared withempty vector control (Fig. 7C). These findings pointed to apivotal role of GRP78 modulation in the tumor-suppressiveeffect of SCNN1B in gastric cancer.

DiscussionIn this study, we identified that SCNN1B is readily expressed in

normal gastric tissues, but is frequently silenced in gastric cancercell lines and primary gastric cancer. Silencing of SCNN1B isassociated with promoter methylation. Demethylation treatmentwith 5-Aza restored expression of SCNN1B, confirming thatpromoter hypermethylation mediates the transcriptional silenc-ing of SCNN1B in gastric cancer.

SCNN1B gene silencing in gastric cancer suggests thatSCNN1B may possess tumor-suppressive function and itsdownregulation may contribute to the development and pro-gression of gastric cancer. Consistent with our hypothesis, theectopic expression of SCNN1B in four gastric cancer cell lines(AGS, BGC823, MGC803, and MKN45) significantly sup-pressed cell proliferation in vitro, while its knockdown in MKN1and NCI-N87 cells, which express endogenous SCNN1B, pro-moted cell viability. Tumor-suppressive effect of SCNN1B wasvalidated in vivo, as evidenced by the diminished growth ofSCNN1B-expressing MKN45 and BGC823 cells in nude mice.SCNN1B suppressed gastric cancer cell proliferation throughapoptosis induction and inhibition of cell-cycle progression.SCNN1B overexpression induced apoptosis in gastric cancercells by activating both intrinsic and extrinsic apoptosis path-ways, leading to the cleavage of caspase-8, caspase-9, and thatof the downstream effectors, caspase-7 and PARP. Moreover,SCNN1B inhibited cell-cycle progression at G0–G1 phase,which was associated with upregulation of p27kip1 and sup-pression of cyclin D1 and CDK2. Cyclin D1/CDK2 forms an

active complex that promotes G1–S transition, while p27kip1

binds to and inhibits cyclin D1/CDK2 activity (18). SCNN1Bhence tips the balance of gene expression toward that of cell-cycle arrest. Inhibition of cell growth in vivo by SCNN1B wasconfirmed by the reduced Ki-67 index in SCNN1B-expressingMKN45 xenografts. Metastasis is a major cause of cancer-relateddeaths. Here, we revealed that SCNN1B functions as a metas-tasis suppressor in gastric cancer by inhibiting cell migrationand invasion, and concomitantly promoting cell adhesion.Taken together, these results indicate that SCNN1B functionsas a tumor suppressor by inhibiting cell growth and metastasisin gastric cancer.

To further elucidate the mechanism of action of SCNN1B, weperformed co-IP and mass spectrometry, which led to theidentification of GRP78 as an interacting partner. The directinteraction between SCNN1B and GRP78 was validated by co-IP in gastric cancer cells and their colocalization by confocalimmunofluorescence microscopy. Moreover, ectopic SCNN1Bexpression reduced the protein expression of GRP78 withoutaltering its mRNA expression. Instead, we showed that SCNN1Bexpression induced polyubiquitination of GRP78, whichtagged the protein for degradation in the proteasome system(19). Reexpression of GRP78 in SCNN1B-expressing gastriccancer cells abrogated the inhibitory effects of SCNN1B on cellproliferation and cell migration, whereas GRP78 knockdownfurther aggravated SCNN1B-mediated growth inhibition. Thesedata indicated that SCNN1B mediates its tumor-suppressiveeffect by regulating GRP78, which is a master regulator of theUPR response. GRP78 is frequently upregulated during cancerprogression to counter UPR, maintain ER homeostasis, andpromote cell survival (15, 16).Taken together, interplaybetween SCNN1B and GRP78 regulates the stability of GRP78,which has serious repercussion on cell survival and migration/invasion.

GRP78 controls the UPR via the sequestration of IRE1a,PERK, and ATF6 (20, 21). While UPR is initiated as a prosurvi-val mechanism, sustained activation of this pathway inducesapoptotic cell death and cell-cycle arrest (22). We demonstratedthat the ectopic expression of SCNN1B, through suppressingGRP78 expression, triggered the proapoptotic arm of the UPRresponse. This was exemplified by the increased PERK expres-sion, which, in turn, induced expression and nuclear localiza-tion of transcription factors ATF4 and CHOP in SCNN1B-expressing gastric cancer cells. Tunicamycin, an UPR inducer,exacerbated the inhibitory effect of SCNN1B on gastric cancercell proliferation, implying that modulation of UPR responseplays an important role in the tumor-suppressive effect ofSCNN1B. ATF4 and CHOP have been shown to induce apo-ptosis following prolonged stress, in part, by increasing proteinload and ATP depletion (23). CHOP also initiates expression ofproapoptotic genes such as DR5 (24), BIM (25), and PUMA(26). UPR induction has also been associated with G1–S cell-cycle arrest via downregulation of cyclin D1 (27) and upregula-tion of p27kip1 (28). GRP78 is also known to promote cancermetastasis, independent of UPR signaling (17). Collectively,our findings suggested that SCNN1B exerts a tumor-suppressiveeffect through its involvement in regulating the expression ofGRP78 and the UPR response signaling pathway.

Finally, we investigated the clinical importance of SCNN1Bexpression and promoter methylation in primary gastric cancer.Using a TMA cohort with 245 primary gastric cancer patients,

Qian et al.





we found that expression of SCNN1B was an independentprognostic factor of favorable patient survival by multivariateCox regression analysis. Moreover, SCNN1B protein expressionwas significantly associated with survival benefit in late-stage(TNM stages III/IV) gastric cancer. This was further validatedby the association of high SCNN1B mRNA expression withimproved survival of late-stage gastric cancer patients in anoth-er two gastric cancer cohorts (29, 30). Gastric cancer variesgreatly in clinical outcome depending on the aggressiveness ofindividual tumors. Currently, TNM staging is clinically themost important predictor of patient survival in gastric cancer,and additional prognostic markers are necessary to provide amore accurate assessment of disease outcomes. SCNN1B wasidentified to be associated with patient outcome, and thereforeour data suggested that SCNN1B may serve as a novel prog-nostic marker for gastric cancer patients. Moreover, SCNN1Bwas found to suppress gastric cancer cell growth; therefore, itmay serve as a therapeutic target of gastric cancer.

In summary, we identified for the first time that SCNN1Bacts as a tumor suppressor through induction of apoptosis andcell-cycle arrest, and inhibition of cell migration and invasion.We also uncovered that SCNN1B exerts its effect by directinteraction with GRP78, which led to its degradation andsubsequent induction of UPR (Fig. 7D). The expression statusof SCNN1B may serve as prognostic markers in primary gastriccancer.

Disclosure of Potential Conflicts of InterestF.K.L. Chan has received speakers' bureau honoraria from AstraZeneca,

Pfizer, Takeda, and is a consultant/advisory board member for Eisai. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Qian, C.C. Wong, F.K.L. Chan, J. YuDevelopment of methodology: J. Xu, Y. Zhang, P.W.Y. ChiuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.C. Wong, H. Chen, W. Kang, H. Wang, L. Zhang,W. Li, M.Y.Y. Go, P.W.Y. Chiu, E.K.W. NgAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Qian, H. Chen, W. LiWriting, review, and/or revision of the manuscript: Y. Qian, C.C. Wong,E.K.W. Ng, F.K.L. Chan, J. YuAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Xu, H. Wang, E.S.H. Chu,M.Y.Y. Go, J.J.Y. Sung, J. YuStudy supervision: C.C. Wong, J. Si, J. Yu

AcknowledgmentsThe authors thank Dr. Zhinong Jiang, Sir Run Run Shaw Hospital (Hang-

zhou, China) and Dr. Ye Cheng, Zhejiang Cancer Hospital (Hangzhou, China).These two pathologists both read and scored the TMA slides of gastric cancerpatients.

Grant SupportThis project was supported by research funds from RGC-GRF (14114615

and 766613) from Hong Kong; Shenzhen Municipal Science and TechnologyR & D Fund (JCYJ20130401151108652), and Shenzhen Virtual UniversityPark Support Scheme to CUHK Shenzhen Research Institute; NationalNatural Science Foundation of China (NSFC; 81502064); and Direct grantfor Research 2013/2014, CUHK (4054100).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 14, 2016; revised November 14, 2016; accepted December 8,2016; published OnlineFirst February 15, 2017.

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2017;77:1968-1982. Published OnlineFirst February 15, 2017.Cancer Res Yun Qian, Chi Chun Wong, Jiaying Xu, et al. Growth and Metastasis via GRP78 DegradationSodium Channel Subunit SCNN1B Suppresses Gastric Cancer

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