Down-regulation Of Calcium-activated Chloride Channel TMEM16A Activates Autophagy In Tamoxifen-resistant Breast Cancer Cells

Objective: Breast cancer is a common malignant tumor in women and a common cause of death from cancer in women.Estrogen receptor(ER)-positive breast cancer is the most common breast cancer,accounting for about 70% of all breast cancers.Tamoxifen is the main drug for endocrine therapy of ER-positive breast cancer.However,approximately30% of patients develop primary or secondary resistance to tamoxifen and cause tumor recurrence.Therefore,in-depth study of the mechanism of tamoxifen resistance in breast cancer and the discovery of new treatment strategies to overcome tamoxifen-resistant in breast cancer are scientific problems that need to be solved urgently.Based on the identification of calcium-activated chloride channel TMEM16 A as the target of tamoxifen,this study verified the new mechanism of tamoxifen inhibiting breast cancer through targeted inhibition of TMEM16 A,and further study the mechanism of tamoxifen resistance mediated by down-regulation of the TMEM16 A channel,and provide new theoretical basis and experimental data for the treatment of breast cancer resistance to tamoxifen.Methods: Patch clamp whole-cell recording was used to record and analyze the effect of4-Hydroxytamoxifen(4-OHT)on the chloride current of TMEM16 A.CCK-8 experiment was used to detect the inhibition of tamoxifen on breast cancer cells proliferation after knocking down TMEM16 A.The Kaplan-Meier method was used to analyze the survival of ER-positive breast cancer patients who received tamoxifen treatment or not with the expression of TMEM16 A.The high-concentration and short-time 4-OHT shock method was used to induce the construction of breast cancer tamoxifen-resistant strains MCF7 R and T47 DR,and the CCK-8 assay,wound healing assay and transwell assay were used to verify the construction of resistant strains.The expression of TMEM16 A m RNA in tamoxifen-resistant cells MCF7 R and T47 DR was detected by q RT-PCR,and western blot was used to detect the expression of TMEM16 A protein in tamoxifen-resistant cells MCF7 R and T47 DR.Western blot was used to detect the expression of TMEM16 A,p62and LC-3 proteins in MCF7 R and T47 DR after overexpression of TMEM16 A.CCK-8assay,wound healing assay and transwell assay were used to detect the proliferation,migration and invasion of MCF7 R and T47 DR after overexpression of TMEM16 A.Results: 4-Hydroxytamoxifen can inhibit the calcium-activated chloride current of TMEM16 A in a dose-dependent manner.Knockdown of TMEM16 A calcium activated chloride channels significantly reduced the effect of tamoxifen on breast cancer cell proliferation.Kaplan Meier survival analysis showed that in patients who did not receive tamoxifen,low TMEM16 A expression was significantly associated with longer overall survival(OS),and conversely,it was associated with shorter OS in patients who received tamoxifen.The proliferation,migration and invasion of breast cancer tamoxifen-resistant cells MCF7 R and T47 DR were enhanced.The relative expression levels of TMEM16 A m RNA and protein in MCF7 R and T47 DR cells were down-regulated,p62 protein expression was down-regulated,and LC3 Ⅱ /I increased.After MCF7 R and T47 DR overexpress TMEM16 A,the expression of p62 protein was up-regulated,and LC3Ⅱ/I decreased.The proliferation,migration and invasion ability of MCF7 R and T47 DR overexpressing TMEM16 A decreased.Conclusion: 4-Hydroxytamoxifen directly inhibits the chloride current of TMEM16 A,and tamoxifen targets TMEM16 A to inhibit breast cancer cell proliferation.TMEM16 A lowexpression is associated with poor prognosis in breast cancer patients following tamoxifen treatment.In breast cancer tamoxifen-resistant cells,the expression of TMEM16 A will be significantly down-regulated,and the autophagy pathway will be significantly activated.After the expression of TMEM16 A in the tamoxifen-resistant cells is restored,autophagy will be suppressed accordingly,and the sensitivity to 4-OHT will be restored.