Simvastatin inhibits oral squamous cell carcinoma by targeting TMEM16A Ca2+-activated chloride channel

Abstract

Purpose: Ca²⁺-activated chloride channel TMEM16A has been found to be overexpressed in many cancers including head and neck squamous cell carcinoma (HNSCC). Nevertheless, the role of TMEM16A in oral squamous cell carcinoma (OSCC) remains unclear. Although simvastatin is known to produce anti-tumor effect, the mechanisms by which simvastatin inhibits cancer remain unclear.

Methods: In this study, we explored the role of TMEM16A expression in human OSCC tissues using both TCGA dataset and immunohistochemistry. CCK-8 assay was applied to evaluate cell proliferation. Patch clamp technique was applied to record TMEM16A Cl^- currents.

Results: We found that high TMEM16A expression is related with large tumor size, lymph node metastasis, and poor clinical outcome in patients with OSCC. In addition, TMEM16A overexpression could promote cell proliferation, and inhibition of TMEM16A channel activities could suppress cell proliferation in OSCC cells. Furthermore, simvastatin could suppress TMEM16A channel activities, and inhibited cell proliferation in OSCC cells via TMEM16A.

Conclusion: Our findings identify a novel anti-tumor mechanism of simvastatin by targeting TMEM16A. Simvastatin may represent an innovative strategy for treating OSCC with high TMEM16A expression.

Keywords: Ca2+-activated chloride channel; Oral squamous cell carcinoma; Simvastatin; TMEM16A.

Fig. 1

High TMEM16A expression is related with poor clinical outcomes in patients with OSCC. a The normalized expression of TMEM16A mRNA in OSCC tissues samples and paired nearby non-tumorous oral tissues samples from TCGA dataset. *p < 0.05. b–d. TMEM16A expression levels in OSCC patients with T1/T2 and T3/T4 stages (b), N0 and N1–N3 stages (c), and stage I/II and stage III/IV (d) from TCGA dataset. *p < 0.05. e Survival curves demonstrate the relationship between TMEM16A expression and overall survival in OSCC patients from TCGA dataset. p < 0.05. f–h. The typical immunohistochemical staining images for the TMEM16A expression in OSCC tissue (g and h) and normal oral mucosa (f) samples. i TMEM16A expression levels in OSCC tissue samples and normal oral mucosae samples according to the immunoreactivity score, *p < 0.05. j–l. TMEM16A expression levels in OSCC patients with T1/T2 and T3/T4 stages (j), N0 and N1–N3 stages (k), and stage I/II and stage III/IV (l) according to the immunohistochemical staining results, *p < 0.05. m Survival curves demonstrate the relationship between TMEM16A expression and overall survival in patients with OSCC according to the immunohistochemical staining results, p < 0.05

Fig. 2

TMEM16A mediates CaCCs in OSCC. a Western blot show TMEM16A protein expression in HaCaT cells and OSCC cell lines. b Western blot show TMEM16A protein expression in transfected CAL-33 cells (scrambled shRNA or TMEM16A-shRNA). c Quantification analysis results of the protein expression of TMEM16A in b. *p < 0.05. d Mean current–voltage (I/V) relationships for traces of currents activated by 0, 0.36, 1 μM Ca²⁺ in CAL-33 cells. e Representative whole-cell currents in d. f Representative currents in transfected CAL-33 cells (1 μM Ca²⁺). g Mean current densities (+ 100 mV) for transfected CAL-33 cells. *p < 0.05. h Time courses of changes of currents (1 μM Ca²⁺) in CAL-33 cells treated with T16Ainh-A01 (20 μM) or DMSO. The arrow represents use of T16Ainh-A01 or DMSO. i, j Representative currents (1 μM Ca²⁺) in CAL-33 cells before and after DMSO (i) or T16Ainh-A01 treatment (j) in h. k Inhibition rates of T16Ainh-A01 and DMSO in h. *p < 0.05

Fig. 3

TMEM16A promotes proliferation in OSCC cells. a CCK-8 shows cell viability in transfected CAL-33 cells. *p < 0.05. b CCK-8 shows cell viability in CAL-33 cells with and without T16Ainh-A01 application (20 μM) for 48 h. *p < 0.05. c Western blot show TMEM16A expression in transfected CAL-27 cells (empty vector or TMEM16A-overexpressing plasmids). d CCK-8 shows cell viability in transfected CAL-27 cells. *p < 0.05

Fig. 4

Simvastatin inhibits TMEM16A channel function. a Time courses of changes of currents (1 μM Ca²⁺) in CAL-33 cells treated with simvastatin (SV) (0.1 μM, 1 μM, 5 μM, 10 μM, 50 μM) and DMSO. The arrow represents application of simvastatin or DMSO. b Inhibition rates of DMSO and simvastatin (0.1 μM, 1 μM, 5 μM, 10 μM, 50 μM) in a. *p < 0.05. c–h Representative currents (1 μM Ca²⁺) in CAL-33 cells before and after DMSO (c) and simvastatin (0.1 μM, d; 1 μM, e; 5 μM, f; 10 μM, g; 50 μM, h treatments in a. i IC₅₀ curve of simvastatin according to b. j Western blot show TMEM16A expression in CAL-33 cells with or without simvastatin treatment (10 μM) for 48 h. k Statistics analysis of TMEM16A protein expression in j

Fig. 5

Simvastatin inhibits proliferation in OSCC cells via TMEM16A. a CCK-8 shows cell viability of HaCaT cells, CAL-27 cells, and CAL-33 cells treated with DMSO or simvastatin (10 μM) for 48 h. *p < 0.05. b CCK-8 shows cell viability of CAL-33 cells treated with DMSO or simvastatin (SV) (1 μM, 10 μM, and 20 μM) for 48 h. *p < 0.05. c CCK-8 shows cell viability in CAL-33 cells treated with DMSO or simvastatin (10 μM) in the presence and absence of MVA (500 μM) for 48 h. *p < 0.05 vs DMSO. ^#p < 0.05 vs DMSO + MVA. d Inhibition rates of simvastatin (10 μM) in c. *p < 0.05. e CCK-8 shows TMEM16A expression in transfected CAL-33 cells in the presence and absence of simvastatin (10 μM) for 48 h. *p < 0.05 vs scrambled shRNA + DMSO. ^#p < 0.05 vs TMEM16A-shRNA + DMSO. f Inhibition rates of simvastatin (10 μM) in e. *p < 0.05. g CCK-8 shows transfected CAL-27 cells with or without simvastatin (10 μM) application for 48 h. *p < 0.05 vs vector + DMSO. ^#p < 0.05 vs TMEM16A OE + DMSO. h Inhibition rates of simvastatin (10 μM) in g. *p < 0.05