Investigation Of The Role Of ITGB1 In Gper-Induced Tamoxifen Resistance And The Underlying Mechanisms In Breast Cancer Cells

Objective:To investigate the role of ITGB1 in tamoxifen resistance initiated by GPER and the relevant mechanisms in breast cancer cells.Methods:The expression of ITGB1 and biomarkers of epithelial-mesenchymal transition (EMT) were evaluated immuno-histochemically in 53 specimens of metastases (MTs) and paired primary tumors (PTs). The expression of ITGB1 was evaluated by Western blot in wild MCF-7 cells and their TAM-resistant (MCF-7R) subclones. GPER agonist G1 and antagonist G15 were used to verify the result of our previous cDNA microarray that expression of ITGB1 was regulated by GPER. Moreover, the specific EGFR inhibitor AG1478, the MAPK/ERK inhibitor U0126 and the PI3K inhibitor were used to investigate the signaling pathway involved in GPER-induced ITGB1 expression. The lentivirus vector containing ITGB1 shRNA was constructed, and then the expression of ITGB1 in MCF-7R cells was silenced by the recombinant lentivirus. The function of ITGB1 was investigated in TAM-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R ITGB1-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blot analysis.Results:ITGB1 and mesenchymal biomarkers (Vimentin and Fibronectin) expression in MTs increased compared to the corresponding PTs; a close expression pattern of ITGB1 and GPER were in MTs. Increased ITGB1 expression was also confirmed in MCF-7R cells compared with MCF-7 cells. Additionally, the expression of ITGB1 was induced by TAM through GPER/EGFR/ERK signaling pathway in MCF-7R cells. Interestingly, silencing of ITGB1 partially rescued the sensitivity of MCF-7R cells to TAM. Importantly, the cell migration and EMT induced by cancer-associated fibroblasts (CAF) were reduced by knockdown of ITGB1 in MCF-7R cells. In addition, the downstream kinases of ITGB1 including focal adhesion kinase (FAK), Src and AKT were activated in MCF-7R cells and maybe involved in the interaction between cancer cells and CAF.Conclusion:GPER/EGFR/ERK signaling upregulates ITGB1 expression and activates downstream kinases, which contributes to CAF-induced cell migration and EMT, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in ITGB1 dependent pattern. Thus, ITGB1 may be a potential target to improve anti-hormone therapy responses in breast cancer patients.