The Role And Underlying Mechanism Of Calcium-activated Chloride Channel Protein ANO1 In The Pathogenesis And Development Of Ovarian Cancer

Ovarian cancer is one of the common gynecological tumors with the highest incidence and mortality rate among epithelial ovarian cancers.According to the pathological classification epithelial ovarian cancer can be mainly divided into four types such as serous ovarian cancer,mucinous ovarian cancer,clear cell carcinoma and endometrial cancer.Although ovarian cancer is known as the"silent killer",more than 80%of the cases are symptomatic and the symptoms are similar to the symptoms of women's physiological,gastrointestinal and urinary diseases,which can easily lead to misdiagnosis.Ovarian cancer cells can be metastasized through peritoneal implantation,blood and lymph.Only 20%of patients can be found when ovarian cancer is confined to ovarian tissue(stage I),in which 90%of patients can get a better prognosis.When the disease affects the pelvic organs(stage II),the abdominal cavity(stage III)and metastasis beyond the peritoneum(stage IV),the cure rate and prognosis of ovarian cancer are gradually reduced.Thus,early diagnosis is essential for treatment of ovarian cancer.Although the application of the tumor marker CA-125 has improved the screening efficiency of ovarian cancer,other peritoneal and pleural diseases can also lead to an increase in CA-125.Therefore,there is a need for new non-invasive,highly sensitive and specific tumor markers in the clinic.Although 70%of postoperative patients are sensitive to drugs platinum and paclitaxel,their resistance also develops,prompting researchers to identify new drug targets for their treatment.As early as the year of 2000,several research groups found that DOG1 gene was highly expressed in tumors.The DOG1 gene wa salso named as TMEM16A,TAOS2,and ORAOV2 in the literature.In 2008,three independent laboratories discovered that DOG1 gene encodes a calcium-activated chloride channel protein,and was renamed to ANO1(anoctamin 1).The ANO1 family is consisted of 10 members,ANO1-10.Among the 10 members,ANO1 and ANO2 are known to function as calcium-activated chloride channels.ANO1 has a protein structure of 10 transmembrane regions and is widely expressed in the glandular secretion epithelium of salivary glands,pancreas,airway epithelium,and also in smooth muscle and sensory neurons.ANO1 functions mainly to mediate transepithelial transport and regulate tracheal fluid secretion,gastrointestinal motility,exocrine gland secretion,renal function and smooth muscle contraction and injury perception.In recent years,more evidence has shown that ANO1 is highly expressed in tumors and can promote tumor development,such as head and neck lymphoma,lung cancer,stomach cancer,gastrointestinal stromal tumor,prostate cancer,oral cancer,pancreatic cancer,breast cancer,et al.However,it is not known whether ANO1 plays a role in the pathogenesis of ovarian cancers.Objective:The main purpose of this study was to explore the role of ANO1 in human epithelial ovarian cancer and its underlying mechanism.Method:Immunohistochemical stainings were performed to evaluate the ANO1 expression in human ovarian cancer tissues.The correlation between ANO1 expression and clinical pathological parameters including gender,pathological differentiation,pathological staging,pathological type was further estimated.The expression levels of ANO1 mRNA in peripheral blood mononuclear cells(PBMCs)from patients with epithelial ovarian cancer or non-cancer patients as control Western-blot assay was used to explore the expression of ANO1 in cell lines of ovarian cancer.SKOV3,Caov-3 cell lines originated from serous ovarian cancer and ES-2 cell line isolated from clear-cell ovarian cancer were used to evaluate ANO1 expression.SiRNA1,siRNA2 and scrambled siRNA were designed for ANO1 silencing experiments.MTT assay was used to measure SKOV3 cell viability.After transfected with siRNA,cells were then incubated with 5-ethynyl-2?-deoxyuridine(EdU)which incorporates into the duplication of SKOV3 DNA.Soft agar colony formation assay was performed for measuring effect of ANO1 on proliferation of SKOV3 cells.Cell migration was assessed using in vitro wound-healing assay.The relative migration area of SKOV3 cells treated with ANO1-siRNA2 or scrambled siRNA was determined.After transfected with siRNA,SKOV3 cells expressing scrambled siRNA or ANO1-siRNA2 in serum free medium were plated in the upper chambers and incubated for 24 hours.Cells migrated to the lower surface were counted and analyzed among the groups of ANO1-siRNA2 and scrambled siRNA.In vivo experiments assessed the effect of silencing ANO1 on the growth of xenografts.The PDX(Patient-derived xenograft)experiment evaluated the effect of silencing ANO1 on the growth of PDX xenografts.Cell confluency and Western blot assays were performed to estimate inhibition effect of ANO1 knockdown on growth of ovarian cancer cell.SKOV3and Caov-3 cells were transfected with scrambled siRNA or ANO1-siRNA2.LY294002,a specific inhibitor for PI3K-Akt signaling,was added into the cells to evaluate its effect on ANO1-mediated promotion of tumor cell growth.Results:1.IHC staining of ANO1 showed that ANO1 was highly expressed in epithelial ovarian cancer tissues(74 cases),as compared with benign tumors(38 cases)or normal tissues(12 cases).ANO1 upregulation was observed in 78.3%of ovarian cancer cases(58/74).In contrast,the percentage of ANO1 positive staining in benign tumors only accounted for 18.4%(7/38)and 0%in normal ovarian tissues(0/12).Statistical analysis indicated that ANO1 protein was significantly overexpressed in epithelial ovarian cancer samples,as compared with benign tumors.In all 74 of epithelial ovarian cancer tissue samples,statistical analysis showed that there was no statistical significance in the scores of ANO1 immunohistochemical staining among the tumor histopathologic types.Based on the FIGO(International Federation of Gynecology and Obstetrics)staging system,ANO1 protein expression was significantly higher in tumor groups with advanced III/IV stage(72.4%,42 out of 58cases)than early I/II stage(27.6%,16 out of 58 cases),confirming the positive correlation between ANO1 expression and ovarian carcinoma grade.However,no significant correlation was observed between ANO1 expression and patient age.2.ANO1 mRNA was highly expressed in PBMCs from serous ovarian cancer patients with an average value of 6.8×10~-33 relative to the expression of housekeeping gene?-actin,as compared with the value of 7.8×10~-55 for non-cancer patients.ANO1mRNA expression level in PBMCs from serous ovarian cancer patients four weeks post-operation for surgical removal of tumors was significantly reduced to a level similar to non-cancer patients in 7 out of 9 cases of serous ovarian cancer patients.3.Western blot analysis indicated that ANO1 proteins were highly expressed in both SKOV3 cells and Caov-3 cells derived from serous ovarian cancer tissue,whereas ES-2cells originated from clear-cell ovarian cancer showed a weaker expression of ANO1.4.Silencing studies were performed on the cell line SKOV3 with a higher expression of ANO1.ANO1-siRNA2,which has the strongest inhibitory effect on ANO1 expression in SKOV3 cells,and Scrambled siRNA without silencing,were selected for the experiment.Effect of ANO1 on ovarian cancer cells at the cellular level by silencing ANO1 gene in SKOV3 cells.5.MTT results indicated that both ANO1-siRNA1 and siRNA2 were effective in silencing ANO1 expression in SKOV3 cells.In MTT assay,SKOV3 cells transfected with ANO1-siRNA resulted in a time-dependent inhibition of cell viability.6.Silencing ANO1 in ANO1-siRNA2 group gave rise to a decrease of EdU positive cells than scrambled siRNA group.7.Transfection with ANO1-siRNA2 resulted in a significant reduction of SKOV3cell colonies in soft agar,as compared with the control group.8.Silencing of endogenous ANO1 delayed the wound-closing of SKOV3 cells wound healing assay,as compared with the control group in which the wound-closing was much faster.9.Silencing of ANO1 by ANO1-siRNA2 resulted in the reduction of SKOV3 cell invasion about 80%in Transwell assay,as compared to the scrambled siRNA group.10.Tumor volumes and weights were recorded at the fourth week when the animals were sacrificed.The injection of PBS or scrambled siRNA caused the development of xenograft tumors with an average volume of 837.5±102.7 mm~3or 860.5±64.3 mm~3,respectively.By contrast,injection of ANO1-siRNA2 resulted in a significant reduction in tumor volume about 69%to 267.5±46.62 mm~3.We also weighted the tumors,and the average weight of tumors in blank group or scrambled siRNA group was 1.7±0.17g or1.8±0.22 g,respectively,whereas the average weight of tumors in ANO1-siRNA2 group was significantly reduced about 65%to 0.63±0.15g,as compared with either blank or scrambled siRNA group.Suppression of ANO1 significantly inhibits the growth of xenograft tumors.11.The immunohistochemical results of the Patient-derived Xenugraph(PDX)experiment showed that ANO1 was expressed in 4 patients with serous ovarian cancer.One patient with OVPF160087 was highly expressed(++).Three patients with OVPF004,OVPF031,and OVPF027 were moderately expressed(++).OVPF004 was randomly selected to construct a PDX model for in vivo experiments.The experimental results show that silencing ANO1 can inhibit the growth of PDX xenografts.12.Western results showed that silencing of ANO1 by transfection of SKOV3 and Caov-3 cells with ANO1-siRNA2 decreased the expression of ANO1 proteins and reduced the phosphorylated Akt.To further confirm the link of ANO1 expression and PI3K-Akt signaling,we utilized the LY294002,a potent inhibitor of PI3K-Akt signaling,and tested its effect on cell confluency.Inhibition of PI3K-Akt signaling by LY294002also resulted in suppression of cell confluency in both SKOV3 and Caov-3 cells,consistent with ANO1 knockdown effect as seen in ANO1-siRNA2.These results demonstrate that inhibition of PI3K-Akt signaling by specific inhibitor LY294002suppressed the growth of ovarian cancer cells promoted by ANO1 overexpression.Conclusions:1.Calcium-activated chloride channel protein ANO1 is highly expressed in human ovarian cancer tissues,blood and ovarian cancer cells,but is not expressed or detected in benign ovarian tumors and normal ovarian tissues,and its expression level and pathology of cancer tissues Staging was positively correlated with the degree of differentiation.The above results indicate that ANO1 is highly expressed in the presence of ovarian cancer.2.Silencing ANO1 can inhibit the proliferation,migration and invasion of ovarian cancer SKOV3 cells and the growth of tumor-bearing mice.The above results indicate that ANO1 is involved in the development of ovarian cancer.3.Inhibition of ANO1 expression in ovarian cancer SKOV3 and Caov-3 cell lines can reduce Akt phosphorylation.Blocking the Akt signaling pathway with the inhibitor LY294002 inhibits the growth of tumor cells by ANO1.The above results indicate that ANO1 acts through the PI3K-Akt pathway,and ANO1 can be used as a target for the prevention and treatment of ovarian cancer.