GNAS Promotes Breast Cancer Cell Proliferation And EMT Via PI3K/AKT/Sanil1/E-cadherin Axis

Breast cancer is the most common malignant tumor in women.According to statistics,the risk of breast cancer in American women is as high as 12% in their lifetime,in another word,one in eight women will suffer from breast cancer.In our country,the incidence of breast cancer is the highest among female.Although with the development and progress of society,people’s living environment and medical conditions have been improved significantly,the incidence of breast cancer is still increasing,and the patients are becoming younger and younger.The incidence and mortality are not effectively controlled,seriously threatening women’s health.Therefore,the study of breast cancer’s development,progression and treatment is more and more urgent.In recent years,studies have found that abnormal expression or mutation of oncogenes,such as Pten,Kras,BRCA1/2 and CDK4/6 etc.are closely related to the occurrence and development of breast cancer.Currently,targeted drugs for breast cancer,such as trastuzumab,pertuzumab,lapatinib,bevacizumab and palbociclib,specifically target Her2,VEGF,CDK4/6,etc.,which can significantly improve the quality of life and prolong the survival time of breast cancer patients.However,the small part of applicable population,high price and drug resistance severely limit the therapeutic effect of breast cancer.Therefore,it is an urgent to explore more new genes closely related to the occurrence and developmentof breast cancer,for revealing the molecular mechanism of breast cancer,and providing new targets for the treatment of breast cancer.G protein is an important cell signal transducer,including alpha,beta and gamma subunits,which binds to many receptors on the cell membrane(G protein coupled receptors).After binding to the corresponding receptors,the alpha subunit of G protein dissociates with the beta and gamma subunits.And then the alpha subunit can transform GTP into GDP,produce the second messenger molecule cAMP,and activate the downstream signaling pathways of protein kinase A(PKA).Although it has been well documented that G protein-coupled receptors are closely related to the occurrence and development of breast cancer,such as GPCR81,PAR1,GPR110,GPR19 and GPER,the relationship between G protein itself and breast cancer is still largely unknown.This study was based on the expression level of GNAS in breast cancer tissue microarray,combined with clinical indicators and follow-up information,systematically explored the correlation between GNAS and clinical prognosis of breast cancer.This study is mainly divided into three parts:PART Ⅰ:EXPRESSION AND CLINICAL SIGNIFICANCE OF GNAS IN BREAST CANCERPurpose:To investigate the expression of GNAS in breast cancer and its relationship with clinical parameters.Methods:Immunohistochemistry(IHC)was used to detect the expression of GNAS in 150 breast cancer tissues,and the expression level was scored.Kaplan-Meier was used to analyze the relationship between GNAS expression and Overall Survival(OS)of patients and clinical indicators.The expression of GNAS in fresh breast cancer/adjacent tissues was detected by immunoblotting(WB).Ki-67 was stained in breast cancer tissues with different GNAS expression levels.Results:The high-and low-expression of GNAS in breast cancer accounted for 64.7%(97/150) and 35.3%(53/150),respectively.The high expression of GNAS was related to distant metastasis(p=0.026) and clinical stage(p=0.001);the overall survival time of breast cancer patients with high expression of GNAS was shorter(p=0.021);the expression level of GNAS in breast cancer tissues was higher than that in adjacent tissues(p=0.021).The proportion of Ki-67 positive cells was higher in GNAS higher breast cancer tissues(p= 0.0351).Conclusion:Breast cancer patients with high GNAS expression have higher clinical stage,more prone to distal metastasis,more proliferative ability and shorter overall survival time.PART Ⅱ:GNAS PROMOTES PROLIFERATION,MIGRATION AND INFILTRATION OF BREAST CANCER CELLSPurpose:To explore the function of GNAS in cancer cell proliferation and migration.Methods:The viability and proliferation of breast cancer cells were detected by CCK8,EdU incorporation and colony formation assays after siGNAS infection;the cell cycle and apoptosis were detected by Flow Cytometry;wound healing and Matrigel assays were used for evaluating cell migration and infiltration;and Orthotopic nude mouse model was used to investigate the effect of GNAS silencing on breast cancer growth in vivo.Results:Decreasing the expression of GNAS decreased the cell viability(p=0.039) and proliferation(p=0.0016) of breast cancer cell line MDA-MB-231,blocked cell cycle and reduced S-phase cells,and decreased the migration and infiltration ability of tumor cells.The decrease of GNAS significantly delayed the growth of breast cancer in nude mice.Conclusion:High expression of GNAS can promote the proliferation,migration and invasion of breast cancer cells in vitro an in vivo.PART Ⅲ:GNAS PROMOTES CANCER CELL PROLIFERATION AND EMT THROUGH PI3K/AKT/E-CADHERIN SIGNALING PATHWAYPurpose:To explore the molecular mechanism of GNAS promoting proliferation,migration and invasion of cancer cells.Methods:WB was used to detect the expression of PI3K/AKT signaling pathway and EMT-related proteins;small molecule inhibitor of PI3K,BKM120 was used to block PI3K/AKT signaling pathway;CCK8,EdU and colony formation assays were used to detect the viability and proliferation of breast cancer cells;wound healing and Matrigel assays were used for evaluating cell migration and infiltration.The expression of p-AKT,E-cadherin and Snail-1 in breast cancer tissues of nude mice were detected.Results:In GNAS-silenced MDA-MB-231 cells,PI3K/AKT signaling pathway was inhibited,with E-cadherin expression increased and snail-1 decreased.And BKM120 reduced the proliferation,migration and invasion of cancer cells the same as effects caused by siGNAS.Conclusion:GNAS promotes cell proliferation and EMT through PI3K/AKT/E-cadherin signaling pathway.