Research On The Relationship Between BAG-1Gene And TAM Response In Breast Cancer

ObjectiveTamoxifen, one of the most commonly used endocrine therapies, is widely considered as a standard treatment for both early and advanced estrogen-and/or progesterone-receptor positive breast cancer. It has been shown to improve relapse-free and overall survival and to reduce the incidence of cotralateral breast cancers. However, data shows that about30-50%of women with ER-positive breast cancer didn’t response to tamoxifen. One hypothytic explaination was raised that certain critical molecules may affect the efficacy of TAM by involving in the ER signaling pathways and interact with ER%PR. BAG-1is a multifunctional protein that interacts with a number of molecules to regulate the diverse biological processes. Studies found that in breast cancer cell lines, BAG-1prevented cells from undergoing apoptosis induced by radiation, chemotherapy and stress. What’s more, some studies reported a strong relationship between the increased BAG-1expression and improved outcome in breast cancer patients treated with TAM. In this study, we tried to assess the association between the expression of BAG-1and the sensitivity to4-OH TAM in the human breast cancer cells, and further investigated the effect of BAG-1on the TAM-induced apoptosis. We hope our finding can prove that BAG-1may serve as a favorable predictive biomarker for the responsiveness to TAM.MethodsBAG-1expression was examined in the MCF-7cells and breast cancer cells that became resistant to4-OH TAM. The4-OH TAM-resistant MCF-7cells were then transfected with the BAG-1siRNA and the4-OH TAM-sensitive MCF-7cells with the plasmids carrying the human BAG-1isoform-specific expression constructs respectively to investigate the effect of BAG-1on the TAM-induced apoptosis.Results1、We established tamoxifen-resistant breast cancer cell line TAMR/MCF-7by continuously exposing to culture medium containing4-OH TAM (10-7M in ethanol) for6months. The acquisition of resistance to TAM in the TAMR/MCF-7cells was confirmed by comparing the morphology、the growth curves、apoptosis rate、cell cycle of the TAMR/MCF-7and MCF-7cells cultured in the presence of4-OH TAM.2、The expression levels of BAG-1in TAMR/MCF-7and MCF-7cells were determined by real-time PCR、RT-PCR and western blot. The result of real-time PCR indicated an almost two-fold increase in BAG-1expression in the TAMR/MCF-7cells compared to that of the MCF-7cells.3、BAG-1expression was dramatically decreased in the TAMR/MCF-7cells transfected with the BAG-1siRNA. We observed significantly increased apoptosis induced by the4-OH TAM in the cells transfected with the BAG-1siRNA compared to that in the control cells. Additionally, the proportion of cells blocked in G0/G1phase in siRNA group was higher than that in control group.4、The expressions of the specific BAG-1isoforms were dramatically enhanced in the MCF-7cells after transfection with each of the isoform-specific constructs. Cells overexpressing BAG-1p50showed significantly less proliferation inhibition、 apoptosis rate than the control group, and the proportion of cells blocked in G0/G1phase in p50group was also significantly fewer then that in control group. However, the cells transfected with the BAG-1p46and p33showed no significant change in response to4-OH TAM treatment.ConclusionOur study indicated that regulations of the BAG-1expression were associated with the altered sensitivity to4-OH TAM in breast cancer cell MCF-7. Distinct isoforms of BAG-1had different anti-apoptotic ability in breast cancer cells treated with the4-OH TAM, and BAG-1p50was the only isoform that inhibited the MCF-7cells from apoptosis induced by TAM. Our finding has significant prognostic, predictive and therapeutic implications in developing future targeted therapy against BAG-1, especially the BAG-1p50.