TMEM16A Ca²⁺-activated Chloride Channel Mediate Inhibitory Effect Of Simvastatin On Breast Cancer

Objective:TMEM16A(also known as Anoctamin1,ANO1)is the molecular basis of calcium-activated chloride channels and has 10 transmembrane domains.TMEM16 A is widely distributed in a variety of cells and participates in the regulation of a variety of important physiological functions.TMEM16 A is highly expressed in a variety of tumors,including breast cancer,and is related to the poor prognosis of patients.Statins(statins)is a first-line lipid-lowering drug commonly used in clinic.it is a hydroxymethyl glutarate monoacyl-Co A(HMG-Co A)reductase inhibitor,which is often used in the treatment of cardiovascular diseases.Statins have inhibitory effects on a variety of tumors,including breast cancer.Compared with hydrophilic statins,lipophilic statins including simvastatin can significantly inhibit the recurrence and metastasis of breast cancer.Previous studies in this experiment showed that there were differences in the ability of lipophilic statin and hydrophilic statin to inhibit TMEM16 A calcium-activated chloride current and breast cancer proliferation.Therefore,the purpose of this study is to explore the inhibitory ability of simvastatin on TMEM16 A and the amino acids that play a key role in binding,to clarify the mechanism of simvastatin inhibiting chloride channel activated by TMEM16 A calcium in breast cancer cells,to find the signal pathway of TMEM16 A promoting the occurrence and development of breast cancer,and to verify the mechanism of simvastatin inhibiting breast cancer through TMEM16A-mediated signal pathway. The purpose of this study is to provide a new idea and experimental basis for the development of drugs targeting TMEM16 A in the treatment of breast cancer.Methods: In this study,CCK-8 cell viability assay was used to detect the proliferation of breast cancer cells treated with simvastatin(simvastatin acid as control),TMEM16 A plasmid was transfected into HEK293 cells,whole-cell patch clamp(Whole cell) recording mode was used to detect and compare calcium-activated chloride currents in HEK293 T cells treated with different concentrations of simvastatin.The amino acids in TMEM16 A were systematically mutated into corresponding amino acids in TMEM16 B by site directed mutagenesis(site-directed mutagenesis).The mutant plasmids of TMEM16 A were amplified and extracted by plasmid transformation and extraction technique.The mutant plasmid containing TMEM16 A was transfected into HEK293 T cells by transient transfection technique,and the whole cell patch clamp technique was used.To detect and compare the calcium activated chloride current of HEK293 T cells transfected with TMEM16 A mutant(WT TMEM16 A as control)before and after simvastatin treatment,and to verify the key amino acids of TMEM16 A binding to simvastatin.To explore the gene enrichment of TMEM16 A in breast cancer through TCGA database and GSEA gene enrichment analysis;to construct the model of TMEM16 A silencing or overexpression of breast cancer cells by knockout TMEM16 A by sh RNA and transfection of TMEM16 A plasmid into MCF-7 and T47 D breast cancer cells;Western blot method was used to detect and compare the phosphorylated expression of EGFR and AKT in TMEM16 A silent and overexpressed breast cancer cells.The phosphorylated expression of EGFR and AKT in breast cancer MCF-7 cells treated with simvastatin was detected.Results: The proliferation of MCF-7 and T47 D cells was detected by CCK-8 cell viability assay.The results showed that simvastatin could significantly inhibit the proliferation of breast cancer MCF-7 and T47 D cells in a concentration-dependent manner.Calcium-activated chloride current was activated by simvastatin in HEK293 T cells transfected with TMEM16 A plasmid,and simvastatin induced TMEM16 A calcium-activated chloride current in a dose-dependent manner.The TMEM16 A mutant was transfected into HEK293 T cells by site-directed mutagenesis,and the changes of calcium-activated chloride current of TMEM16 A mutant were recorded by whole-cell patch clamp(Whole cell).The results showed that R429 A,K430L and R437 F decreased the inhibition of calcium-activated chloride current induced by simvastatin in TMEM16 A,and R429A/K430L/R437 F also decreased the inhibition of calcium-activated chloride current induced by simvastatin.The expression levels of phosphorylated EGFR and AKT of MCF-7 and T47 D were detected by Western blot detection of TMEM16 A overexpression plasmid and Sh RNA knockout plasmid.The results showed that the expression of phosphorylated EGFR and AKT protein was significantly increased in transfected TMEM16 A cells,suggesting that the high expression of TMEM16 A may activate EGFR/AKT signaling pathway.The expression of phosphorylated EGFR/AKT in breast cancer T47 D cells decreased after simvastatin treatment.Conclusion: We found that simvastatin inhibited the proliferation of breast cancer cells and inhibited the activation of chloride channels by TMEM16 A calcium in a dose-dependent manner.The key amino acids for direct binding of simvastatin to TMEM16 A were identified as R429 A,K430L and R437 F.The results showed that TMEM16 A calcium-activated chloride channel could activate EGFR/AKT signal pathway in breast cancer cells,the expression of phosphorylated EGFR/AKT protein in breast cancer cells with overexpression of TMEM16 A was significantly increased,and the expression level of phosphorylated protein of EGFR and AKT decreased after TMEM16 A silencing.TMEM16 A calcium-activated chloride channel function can promote the occurrence and development of breast cancer.Studies have shown that simvastatin inhibits the expression of phosphorylated EGFR and AKT in breast cancer cells.