TMEM16A Ca²⁺-activated Chlorine Channels Promote The Pathogenesis Of Breast Cancer

PartⅠProgesterone promotes the development of breast cancer by upregulating TMEM16A Calcium-activated Chlorine Channels through progesterone receptorObjective:To investigate the effect of calcium-activated chlorine channel on PR signaling pathway of breast cancer.Methods:The expression differences of TMEM16A in PR positive and PR negative human breast cancer tissues were detected by TCGA database and immunohistochemistry.Western blot was used to detect the expression of TMEM16A in MCF-7 cell lines treated with progesterone at different concentrations（0.1-100n M）for 0h-24h and combined progesterone and mifepristone for 24h.The interaction between PR and TMEM16A promoter region was detected by Dual-luciferase reporter system.MCF-7 was transfected with TMEM16A overexpressed plasmid or TMEM16A-sh RNA to obtain cell lines with high or low TMEM16A expression.Western blot was used to detect the expression of PR-B and PR-A proteins in MCF-7 cells with different TMEM16A expression levels.CCK-8 method was used to detect the proliferation of MCF-7 cells with different TMEM16A expression levels and the proliferation of MCF-7 cells treated with progesterone after TMEM16A was knocked out.Western blot was used to detect the expression of TMEM16A after transfection with TMEM16A-OE plasmid and treatment with STAT3 inhibitor for 24h.These results were validated on another breast cancer cell line at T47D.Patch-clamp technique was used to record the T47D cell line administered with different internal calcium stimuli.We recorded calcium-activated chloride current after knocking out TMEM16A on T47D cells,also recorded cells pretreatment with progesterone for 24h and then treated with TMEM16A channel inhibitor T16Ainh-A01（20μM）.Western blot and CCK-8 assay were used to detect the expression of total proteins and phosphorylated proteins of EGFR and STAT3 and cell proliferation after treated with TMEM16A channel inhibitor T16Ainh-A01,respectively.Western blot was used to detect the expression of TMEM16A,PR-B,PR-A,EGFR and STAT3 total proteins and phosphorylated proteins in breast cancer cells transfected with△₄₄₄EEEEEAVKD₄₅₂mutants.Results:High expression of TMEM16A was more frequent in PR-positive breast cancer samples than in PR-negative breast cancer samples.Progesterone increased theexpression of TMEM16A through the interaction between PR and the promoter region ofTMEM16A.TMEM16A overexpression increases the expression of PR-B by activating the EGFR/STAT3 signaling pathway.Mutual activation of TMEM16A and PR-B mediates progesterone-induced cell proliferation.The T47D cell line has calcium-activated chlorine current,which is calcium dependent and voltage dependent.After progesterone pretreatment for 24h,the calcium activated chlorine current in the cells increased,while the calcium activated chlorine current gradually decreased after the acute dosage of T16Ainh-A01.Functional inhibition of TMEM16A channel reduces EGFR/STAT3 signaling and progesterone-induced cell proliferation.TMEM16A channel function inhibition in breast cancer cells transfected with△₄₄₄EEEEEAVKD₄₅₂ mutant reduced the expression of EGFR/STAT3 signaling pathway and decreased the expression of PR-B.Conclusion:Progesterone up-regulates the calcium-activated chloride channel by binding the progesterone nuclear receptor to the TMEM16A promoter region and promotes calcium-activated chloride current.TMEM16A promotes the expression of PR-B in breast cancer cells through the EGFR/STAT3 signaling pathway.TMEM16A mediates PR to promote breast cancer cell proliferation.TMEM16A and PR-B activate each other to form a positive feedback loop.The enhancement of TMEM16A channel activity mediates progesterone to promote breast cancer cell proliferation.PartⅡMoesin regulates the function of TMEM16A channel to promote breastcancer cell proliferationObjective:To explore the mechanism of moesin participating in gate regulation and downstream signal transduction of TMEM16A channel.Methods:TMEM16A currents activated by calcium stimulation in 0.36μM,1μM,25μM and 126μM were collected from T47D cells transfected with moesin overexpression or empty vector plasmid by whole-cell patch clamp technique.We Used Western blot and CCK-8 to detect TMEM16A,p-EGFR,EGFR,p-STAT3,STAT3 protein expression and cell proliferation in T47D cells transfected with TMEM16A over-expression plasmid.We Used Western blot and CCK-8 to detect p-EGFR,EGFR,p-STAT3,STAT3 protein expression and cell proliferation in T47D cells transfected with TMEM16A over-expression plasmid or T16A OE plasmid+sh RNA moesin plasmids.Results:TMEM16A current increased in T47D cells transfected with moesin over-expression plasmid,and the sensitivity of TMEM16A Ca²⁺increased by approximately 10 times.EC50 was 29.37μM in the control group and 3.20μM in the moesin over-expression group.After transfected with moesin over-expression plasmid,the activation current of TMEM16A was fitted into two terms,including fast and slow components,while the activation current of TMEM16A in control group was fitted into one term.Consistent with our previous findings,overexpression of TMEM16A led to increased expression of phosphorylated EGFR and STAT3 phosphorylated proteins and promoted T47D cell proliferation.Transfection with sh RNA-moesin plasmids inhibited EGFR/STAT3 activation and inhibited TMEM16A-induced T47D cell proliferation induced by TMEM16A overexpression.Conclusion:Moesin promotes Ca²⁺sensitivity and activation kinetics of TMEM16A channel in T47D cells.Moesin mediated TMEM16A overexpression induced increased T47D cell proliferation through the EGFR/STAT3 signaling pathway.PartⅢMechanism of TMEM16A high expression promoting breast cancer invasion and metastasis through EGFR/STAT3/ROCK1Objective:To study the mechanism of high expression of TMEM16A in promoting breast cancer invasion and metastasis.Methods:TCGA database was used to analyze the effects of high and low expression of TMEM16A on tumor size（T）,lymph node metastasis（N）,stage of breast cancer,and overall survival of breast cancer patients.Immunohistochemical method was used to analyze the effect of TMEM16A expression on lymph node metastasis.The migration and invasion of breast cancer cells with different TMEM16A expression levels was verified by wound healing assay and Transwell assay.The metastatic tumor model of nude mice was established and HE staining was used to verify the tumorigenesis of breast cancer cells with different TMEM16A expression levels.TCGA database was used to analyze the correlation between TMEM16A and ROCK1,and the effect of TMEM16A and ROCK1 combined expression on lymph node metastasis,the stage of breast cancer,and overall survival of breast cancer patients.Western blot was used to detect the expression of ROCK1 in MCF-7 breast cancer cells transfected with TMEM16A-OE and TMEM16A-sh RNA plasmid.The migration and invasion of MCF-7 breast cancer cells treated with ROCK1 inhibitor Y-27632 was verified by wound healing assay and Transwell assay.The correlation between EGFR and ROCK1 was analyzed by TCGA database.Western blot was used to examine the expression of ROCK1 in MCF-7 breast cancer cells after EGF was given,as well as the expression of downstream pathway proteins and ROCK1 after EGFR inhibitor gefitinib,STAT3 inhibitor JSI-124 were given.The ability of MCF-7 breast cancer cells to invade and migrate was tested by wound healing assay and Transwell test after treatment with gefitinib or JSI-124.These results were confirmed in another breast cancer cell line,T47D.Patch clamp technique was used to record the change of calcium-activated chlorine current of TMEM16A in T47D cells after the administration of T16Ainh-A01.Using Transwell test verified the change of invasion ability of breast cancer cells after T16Ainh-A01 was given or△₄₄₄EEEEEAVKD₄₅₂mutation was transfected.Results:Breast cancer patients with high TMEM16A expression had larger tumors,more lymph node involvement,more severe clinical staging of breast cancer,and shorter survival than patients with low TMEM16A expression.High expression of TMEM16A promoted invasion and migration of breast cancer cells,and nude mice injected with high TMEM16A expression of MCF-7 cells in tail vein showed more lung tumors.ROCK1was positively correlated with TMEM16A.Breast cancer patients with high TMEM16A and ROCK1 expression had more lymph node involvement,more severe clinical staging of breast cancer,and shorter survival than patients with low TMEM16A and ROCK1expression.In MCF-7 cell lines,high expression of TMEM16A promoted high expression of ROCK1.The administration of Y-27632 inhibited the invasion and migration of breast cancer cells.EGFR was positively correlated with ROCK1,and EGF stimulated ROCK1 expression.Gefitinib and JSI-124 inhibited the expression of downstream pathway proteins and ROCK1,and inhibited the invasion and migration of breast cancer cells.These results were also replicated in the T47D breast cancer cell lines.The administration of T16Ainh-A01 inhibited the calcium-activated chlorine current of TMEM16A and the invasion of breast cancer cells.The expression of ROCK1 protein was decreased in breast cancer cells transfected with the plasmid△₄₄₄EEEEEAVKD₄₅₂,and the invasion ability was weakened.Conclusion:TMEM16A promotes invasion and migration of breast cancer cells through the EGFR/STAT3/ROCK1 signaling pathway.Enhancing the channel activity of TMEM16A calcium-activated chlorine channel promotes the invasion of breast cancer cells.