Simvastatin Inhibits Proliferation Of Oral Squamous Cell Carcinoma By Targeting TMEM16A Ca²⁺-activated Chloride Channel

Objective: Oral squamous cell carcinoma（OSCC）is one of the most prevalent cancers in the head and neck region.The current management of OSCC involves a multimodality treatment with surgery,radiation therapy and/or chemotherapy.Despite remarkable improvements in therapy,the five-year survival rate of OSCC remains approximately 50% over the last decade.Identification of a novel biomarker that is important for early diagnosis or targeted therapeutic interventions is of great value for the treatment of OSCC.TMEM16 A has been discovered recently as Ca²⁺-activated chloride channel.It is extensively expressed in many normal tissues and involved in various physiological functions.The dysfunction of TMEM16 A is associated with many diseases including various cancers.TMEM16 A has been reported to be overexpressed in many cancers including head and neck squamous cell carcinoma（HNSCC）.Overexpression of TMEM16 A can be involved with the genesis and development of HNSCC.OSCC is one of the most important part of HNSCC.Nevertheless,the role of TMEM16 A in OSCC remains unclear.Simvastatin is widely used in clinical practice as cholesterol-lowering drug by inhibiting hydroxy-3-methylglutaryl-Co A（HMG-Co A）reductase.Growing evidence has been shown that simvastatin exhibits anti-tumor effects.It has been reported that simvastatin could inhibit cancer cells by MVA-dependent mechanisms and MVA-independent mechanisms.However,detailed molecular mechanism of the oncoprotective activity of simvastatin has not been firmly established.In this study,we aimed to investigate the expression and clinical value of TMEM16 A in OSCC,to investigate the effect of TMEM16 A on proliferation of OSCC,and to investigate the effect of simvastatin on proliferation of OSCC and its molecular mechanism.Methods: RNA sequencing（RNA-Seq）data and clinical data from 305 OSCC tissues and 30 adjacent non-tumorous oral tissue samples were obtained from TCGA database.RNA sequencing（RNA-Seq）data from 40 paired OSCC tissues and adjacent non-tumorous oral tissue samples were obtained from GEO database.84 OSCC tissue samples and 12 specimens of normal oral mucosa were performed with immunohistochemistry staining.The difference of TMEM16 A expression between OSCC tissues and non-tumorous oral tissues and the association of TMEM16 A expression with clinicopathological characteristics of OSCC patients were evaluated.Whole cell patch clamp and current clamp recordings were carried out to record TMEM16 A Ca²⁺-activated Cl-currents（Ca CCs）in CAL-33 cells.CCK-8 assay was applied to identify the proliferation-promoting effect of TMEM16 A in OSCC cells.Whole cell patch clamp and current clamp recordings were carried out to investigate the effect of simvastatin on Ca CCs in CAL-33 cells.Western blot analysis was applied to examine TMEM16 A expression in CAL-33 cells before and after simvastatin treatment.CCK-8 assay was applied to identify the proliferation-inhibiting effect of simvastatin in CAL-27,CAL-33 and Ha Cat cells.CCK-8 assay was applied to identify the effect of MVA,TMEM16 A knock down,and TMEM16 A overexpression on proliferation-inhibiting effect of simvastatin in OSCC cells.Results: The mRNA expression level of TMEM16 A was significantly higher in OSCC samples compared with their nearby non-tumorous oral tissues.The mRNA expression levels of TMEM16 A were significantly related with tumor size,lymph node metastasis,and clinical stage in OSCC patients.The high expression levels of TMEM16 A were more associated with tumors with T3 and T4 stage,N1-N3 stage,and stage III and stage IV.Furthermore,Kaplan-Meier survival analysis demonstrated that high expression of TMEM16 A was significantly related with shorter overall survival in OSCC patients.TMEM16 A protein expression level was significantly higher in OSCC samples compared with normal oral mucosae.Besides,the protein expression levels of TMEM16 A were significantly related with tumor size,lymph node metastasis,and clinical stage in OSCC patients.The high expression levels of TMEM16 A were more associated with tumors with T3 and T4 stage,N1-N3 stage patients,and stage III and stage IV.Furthermore,survival analysis showed that patients with high TMEM16 A expression had significantly reduced overall survival compared with patients with low TMEM16 A expression.TMEM16 A mediates Ca CCs in CAL-33 cells.TMEM16 A knockdown significantly suppressed cell proliferation in CAL-33 cells.T16Ainh-A01（20 μM）also significantly suppressed cell proliferation in CAL-33 cells.TMEM16 A overexpression significantly promoted cell proliferation in CAL-27 cells.Simvastatin significantly inhibited Ca CCs in CAL-33 cells in a dose-dependent fashion.The IC50 of simvastatin was 5.605 ± 1.512 μM.TMEM16 A expression in CAL-33 cells was not significantly altered after simvastatin（10 μM）treatment.Simvastatin treatment significantly inhibited proliferation in OSCC cells CAL-27 and CAL-33,but not in Ha Cat cells.Meanwhile,simvastatin inhibited proliferation in CAL-33 cells in a dose-dependent fashion.MVA（500 μM）could reduce the inhibitory influence of simvastatin on cell proliferation in CAL-33 cells.TMEM16 A knockdown significantly reduced the inhibitory influence of simvastatin on cell proliferation.TMME16 A overexpression increased the sensitivity of CAL-27 cells to simvastatin.Conclusion: TMEM16 A is overexpression in OSCC.High TMEM16 A expression is related with large tumor size,lymph node metastasis and poor clinical outcome in patients with OSCC.In addition,TMEM16 A promoted proliferation of OSCC cells.Furthermore,simvastatin could suppress TMEM16 A channel activities,and inhibited cell proliferation in OSCC cells via TMEM16 A.