TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-β signaling

Abstract

TMEM16A is a newly identified calcium activated chloride channel, and has been reported to be overexpressed by various solid malignant cancers to promote proliferation and invasion, yet little is known about its role in gastric cancer(GC). Therefore, we investigated the role of TMEM16A in GC and its clinical significance by a retrospective analysis of 367 GC patients, and in vitro study was performed for validation and underlying molecular mechanism.TMEM16A was significantly upregulated and amplified in GC tissues, and its overexpression was positively correlated with disease stage, negatively with patient survival and identified as an independent prognostic factor for patient outcome. A negative correlation between TMEM16A and E-cadherin was found in 367 GC specimens. TMEM16A silencing significantly decreased calcium activated chloride currents, impaired TGF-β secretion, reduced E-cadherin expression, and inhibited the migration and invasion without affecting proliferation of GC cells (AGS and BGC-823). Supplement of TGF-β reverted the effects of TMEM16A silencing on E-cadherin expression, cell migration and invasion.In conclusion, TMEM16A promotes invasion and metastasis in GC, and might be a novel prognostic biomarker and potential therapeutic target in the treatment of GC.

Keywords: TGF-β; TMEM16A; gastric cancer; invasion; prognosis.

Figure 1. The expression of TMEM16A protein in gastric cancer, adjacent and normal tissues

A. A representative hematoxylin and eosin (HE) stained mounted section containing gastric cancer, adjacent and normal gastric mucosa (the upper row, original magnification, ×4) with enlarged view(the middle row, original magnification, ×40). The lower row displayed that TMEM16A was strongly expressed in gastric cancer(the right panel), whereas was moderately or weakly expressed in adjacent(the middle panel) and normal tissues(the left panel) (original magnification, ×40). B. Western blot analysis of TMEM16A protein expression in four pairs of matched gastric tumor (T) and adjacent non-tumor mucosa (N). Equal loading of protein was determined by β-actin.

Figure 2. Kaplan–Meier estimated of overall survival according to TMEM16A expression in patients with gastric cancer (log-rank test)

High expression of TMEM16A protein was closely correlated with inferior overall survival(OS) in both testing set A. and overall patients B. (testing set p = 0.022; overall patients p = 0.018).

Figure 3. Evaluation of TMEM16A amplification and overexpression in GC tissues

A. Positive(case 47) and B. negative (case 203) TMEM16A amplification evaluated by FISH analysis. C. High (case 47) and D. low (case 203) TMEM16A expression evaluated by immunohistochemical analysis. Red signals: TMEM16A genes, green signals: chromosome 11 centromere.

Figure 4. The expression of TMEM16A in GC cell lines by western blot analysis

TMEM16A expression was higher in GC cells than that in GES-1, and obviously higher in AGS and BGC-823 cells than that in other GC cells. Equal loading of protein was determined by β-actin.

Figure 5. Knockdown of TMEM16A does not affect AGS cell proliferation

A. BrdU incorporation assay at 2 hours, 4 hours and 8 hours and B. cell count experiments at 24 hours, 48 hours and 72 hours from CTR group, SCR group and ShTM group (n = 6, p > 0.05).

Figure 6. Knockdown of TMEM16A prevents AGS cells migration and invasion

A. Wound closure assay as shown in phase contrast microscopy images. After 48 h, the wound was nearly closed in CTR and SCR groups. However, knockdown of TMEM16A delayed wound closure significantly though an obvious proliferation was seen in all three groups (n = 6, *p < 0.05 vs CTR group). B. In the transwell assay, migrated cells significantly reduced after TMEM16A knockdown (n = 6, *p < 0.05 vs CTR group). C. In the transwell invasion assay, invasiveness was quantified by cells through Matigel and it was showed fewer cells in ShTM group than CTR group and SCR group (n = 6, *p < 0.05 vs CTR group). CTR: control AGS, SCR: scrambled AGS, ShTM: shTMEM16A AGS.

Figure 7. TMEM16A represses E-Cadherin expression

A. Representative images of TMEM16A and E-cadherin immunostaining in gastric cancer, lymphnode metastasis lesion and non-tumor mucosal tissue (case 125). High expression of TMEM16A protein was detected in gastric cancer (n = 367) and lymphnode metastasis lesion (n = 30), and low expression was observed in non-tumor mucosal tissue (n = 20), but corresponding E-cadherin expression demonstrated the converse results. In panel TMEM16A and E-cadherin, the right panels displayed representative TMEM16A and E-cadherin proteins expression in selected zone with enlarged view. B. Knockdown TMEM16A upregulated E-cadherin expression in AGS cells by western blot analysis. Equal loading of protein was determined by β-actin (n = 6, p < 0.05).

Figure 8. TMEM16A represses E-cadherin expression by modulating the secretion of TGF-β1 and TGF-β2

A. Serum levels of TGF-β1 and TGF-β2 was measured by ELISA. Concentrations of TGF-β1 and TGF-β2 were obviously higher in patients with high TMEM16A (n = 62) and low TMEM16A expression(n = 43) than in healthy volunteers(CTR). B. Knockdown of TMEM16A did not affect mRNA expression of TGF-β1 and TGF-β2 in AGS cells(n = 6, p > 0.05). C. Protein expression of TGF-β1 and TGF-β2 in AGS cells increased when knockdown of TMEM16A as shown by western blot analysis(n = 6, p < 0.05). D. Representative Cl⁻ current traces i) and I-V curves ii) of calcium-activated Cl⁻ currents in AGS cells from CTR(n = 7), SCR(n = 5) and ShTM (n = 7) groups (*p < 0.05 vs CTR group). E. Supernatant concentrations of TGF-β1 and TGF-β2 dramatically reduced when knockdown of TMEM16A as shown by ELISA(n = 6, #p < 0.01 vs CTR group, *p < 0.05 vs CTR group). F. Adding recombinant purified TGF-β (rTGF-β; R&D Systems) increased E-cadherin expression in AGS cells with TMEM16A knockdown (n = 6). G. Supplement of recombinant purified TGF-β increased the migration and invasion of AGS cells with TMEM16A knockdown (n = 6). CTR: control AGS, SCR: scrambled AGS, ShTM: shTMEM16A AGS.