TRPC1 Inhibits Cell Proliferation/Invasion and Is Predictive of a Better Prognosis of Esophageal Squamous Cell Carcinoma

Abstract

Background and objectives: In China, over 90% of esophageal cancer (EC) cases are esophageal squamous cell carcinoma (ESCC). ESCC is a frequently malignant tumor with poor prognosis despite the development of comprehensive therapeutic strategies, for which there is still a lack of effective prognostic factors. Previous studies found that the abnormal expression of TRPC1 is closely related to the proliferation, invasion, metastasis, and differentiation of various tumors. However, the relationship between TRPC1 and ESCC is currently unclear. The present study aimed to clarify the clinical significance of TRPC1 and to preliminarily assess the molecular mechanism by which TRPC1 regulates cell proliferation, migration, and invasion in ESCC.

Materials and methods: Immunohistochemistry (IHC) was used to determine the expression of TRPC1 and Ki-67 in 165 cases of ESCC. The correlations between TRPC1 expression and clinicopathological characteristics were determined, and both univariate and multivariate analyses were utilized to quantify the impact of TRPC1 expression on patient survival. Cell Counting Kit-8, scratch wound healing, and transwell assays were used to determine the effects of TRPC1 on proliferation, migration, and invasion in ESCC in vitro, respectively.

Results: The positive expression rate of TRPC1 showed significantly decreased in ESCC (45.50%) compared with the levels in normal esophageal mucosa (NEM; 80.80%) and high-grade intraepithelial neoplasia (HGIEN; 63.20%) (P<0.001). Higher expression rate of TRPC1 was associated with low lymph node metastasis (P<0.001), high differentiation (r_s = 0.232, P=0.003), and low Ki-67 (r_s = -0.492, P<0.001). We further revealed that low expression of TRPC1 was associated with poor prognosis (Disease-free survival, DFS: 95% CI=0.545-0.845, P=0.001; Overall survival, OS: 95% CI=0.553-0.891, P=0.004). Furthermore, we showed that downregulation of TRPC1 promoted the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line EC9706 in vitro. In contrast, overexpression of TRPC1 inhibited the proliferation, migration, and invasion of human esophageal squamous cell carcinoma cell line KYSE150 (P<0.01), in a manner at least in part mediated through the AKT/p27 pathway.

Conclusion: TRPC1 inhibited the proliferation, migration, and invasion of EC9706 and KYSE150 cells, at least, in part mediated through the AKT/p27 pathway in vitro. The downregulation of TRPC1 may be one of the most important molecular events in the malignant progression of ESCC. TRPC1 could be a new candidate tumor suppressor gene and a new prognostic factor of ESCC.

Keywords: TRPC1; cell proliferation; esophageal squamous cell carcinoma; migration and invasion; prognosis.

Figure 1

TRPC1 expression and its relationship with Ki-67. (A) TRPC1-negative expression in NEM. (B) TRPC1-negative expression in ESCC. (C) TRPC1 staining in NEM. (D) TRPC1 was associated with low Ki-67. (E) TRPC1-positive expression in NEM. (F) TRPC1-positive expression in ESCC. (G, H) TRPC1 staining in ESCC, and tumor without TRPC1 staining showed increased Ki-67. (I–L) HE staining was used to observe the histological morphology of NEM (I), TATC (J), HGIEN (K), and ESCC (L) in the same patient. (M–P) IHC staining showed different expression levels of TRPC1 in the same patient among NEM (M), TATC (N), HGIEN (O), and ESCC (P) (magnification: 100×). The scale bar represents 200 μm. NEM, normal esophageal mucosa; TATC, tissue adjacent to carcinoma; HGIEN, high-grade intraepithelial neoplasia; ESCC, esophageal squamous cell carcinoma.

Figure 2

Survival curve of TRPC1 expression in esophageal squamous cell carcinoma patients. (A) Overexpression of TRPC1 predicts better disease-free survival in patients with ESCC. (B) Overexpression of TRPC1 predicts better overall survival in patients with ESCC.

Figure 3

Knockdown of TRPC1 by transfection with siRNA promoted the proliferation, wound healing, migration, and invasion abilities of EC9706 cells. (A) Western blotting showed TRPC1 expression in four esophageal squamous carcinoma cell lines: Eca109, EC9706, KYSE510, and KYSE150. (B) Representative western blot showing the effect of siRNA directed against TRPC1 on the level of TRPC1 protein in EC9706. (C) Analysis of proliferation of cells transfected with siTRPC1 by CCK-8 assay. Cell proliferation was measured 24, 48, 72, and 96h post-transfection. After treatment with siTRPC1, the viability of EC9706 cells was significantly increased. (D) Cellular wound healing after knockdown of TRPC1 in EC9706 cells (magnification: 100×). The rate of wound healing of EC9706-siTRPC1-1 or EC9706-siTRPC1-2 cells was significantly higher than that of EC9706-siNC cells (P<0.01). (E) Cell migration and invasion after knockdown of TRPC1 (magnification: 100×). The cells of the silenced expression group (EC9706-siTRPC1-1 or EC9706-siTRPC1-2) had higher migration and invasion abilities than those of the control group (EC9706-siNC), (P<0.05). NC represented as negative control. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 4

Knockdown of TRPC1 by transfection with shRNA increased the proliferation, wound healing, migration, and invasion abilities of EC9706 cells. (A) Representative western blotting showed the effect of shRNA directed against TRPC1 on the level of TRPC1 protein and revealed that knockdown of TRPC1 induced the phosphorylation of AKT (Ser473), which promoted p27 in EC9706, while total AKT remained unchanged. (B) Analysis of proliferation of cells transfected with shTRPC1 by CCK-8 assay. Cell proliferation was measured 24, 48, and 72 h post-transfection. After treatment with shTRPC1, the viability of EC9706 cells was significantly increased. (C) Wound healing after knockdown of TRPC1 in EC9706 cells (magnification: 100×). The rate of wound healing of the EC9706-shTRPC1-1 or EC9706-shTRPC1-2 cells was significantly higher than that of the EC9706-shNC cells (P<0.01). (D) Cell migration and invasion after knockdown of TRPC1 (magnification: 100×). The cells of the silenced expression group (EC9706-shTRPC1-1 or EC9706-shTRPC1-2) had higher migration and invasion abilities than those of the control group (EC9706-shNC). NC represented as negative control. **P < 0.01, ***P < 0.001,****P < 0.0001.

Figure 5

Ectopic overexpression of TRPC1 inhibited the proliferation, wound healing, migration, and invasion abilities of KYSE150 cells. (A) Ectopic overexpression of TRPC1 decreased the level of phosphorylated AKT (Ser473) and promoted the expression of p27. (B) TRPC1 overexpression inhibited the proliferation of KYSE150 cells, as determined by CCK8 assay. (C, D) Overexpression of TRPC1 suppressed cell migration and invasion, as determined by wound healing and transwell assays, in KYSE150 cells (magnification: 100×). NC represented as negative control. **P < 0.01, ***P < 0.001,****P < 0.0001.