Estrogen Receptor α 66 Mediates Breast Cancer Cell Resistance To Paclitaxel

Breast cancer is the most commonly diagnosed cancer type in women. American Cancer Sociaty estimates, breast cancer has been the highest incidence of femal malignant tumor and ranks the second in female cancer deaths. With the development of economy, in our country, breast cancer has ranked first in female cancer incidences. So more understandings about breast cancer are needed for the improvement of early detection and comprehensive treatment.The expression status of estrogen receptor (ER) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response. There are two main subtypes of ER, ERa and ERβ. Due to the existence of splice variants, ERa has three subtypes, namely ERa66, ERa46 and ERa36, ERa66 is simply called ER66. Data from clinical trials or retrospective analyses suggested that ER66 status might also affect the efficacy of chemotherapy, Specifically, it has been observed that some chemotherapeutic agents may be less effective in patients with ER66+breast tumors than those with ER66-breast tumors. Taxanes (paclitaxel and docetaxel) is an active agent used in breast cancer chemotherapy. Evidence is accumulating that improvements in taxane-based adjuvant chemotherapy disproportionately benefit patients with ER66-breast tumors. In the present study, we investigate paclitaxel susceptibility difference in BC-ER and BC-V cells and its underlying mechanism. The details are listed below:1. Stable transfection of breast cancer cellsWe established several isogenic ER66 cell lines by stable transfection of ER66 expression vectors and empty vector into ER66-Bcap37 breast cancer cells, The expression vector was successfully transfected into Bcap37 breast cancer cells and the expression of ER66 protein was confirmed by RT-PCR and Western-blot, and established Bcap37 transfected with pIRES-ER66 expression vector (BC-ER), or empty vector (BC-V) cells.2. To investigate paclitaxel susceptibility difference in BC-ER and BC-V cellsTo investigate the possible influence of ER66 on the therapeutic efficacy of paclitaxel in BC-ER and BC-V cells. We did MTT assays and Flow cytometric analyses to determine whether the expression of ER66 would affect the sensitivity of Bcap37 cells to paclitaxel. We showed that E2 significantly reduces the overall cytotoxicity of paclitaxel in Bcap37-expressing ER66 but has no influence on the ER66-parental cells.3. To investigate the mechanisms underlying paclitaxel susceptibility difference in BC-ER and BC-V cells(1) Cell proliferation rate was examined to investigate the paclitaxel susceptibility difference in BC-ER and BC-V cellsChemotherapy susceptibility is associated with cell proliferation rate, High growth rate tumors are sensitivity to chemotherapy. MTT assays show E2 would affect the vitality of BC-ER cells, next, we examine the possible effect of E2 on Cell cycle and cyclinD1 protein, the results show E2 decreases the population of cells at G2/S phase and the protein levels of cyclin D1, so E2 can restrain proliferation rate of BC-ER cell.To infer cell proliferation rate decreasesing via E2 in BC-ER cells is likely to strongly influence variation the efficacy of paclitaxel therapy;(2) Apoptosis-associated proteins were examined to investigate the paclitaxel susceptibility difference in BC-ER and BC-V cellsThe data mentioned above reveal that E2 significantly reduces the overall cytotoxicity of paclitaxel in Bcap37-expressing ER66, next, we examined the possible inhibitory effect of ER66 on the ability of paclitaxel to induce breast cancer cell apoptosis. Western-blot exames paclitaxel induce caspase3 protein expressing difference in BC-ER and BC-V cells. The result shows, in BC-ER cells, pretreatment with E2 attenuated the paclitaxel-induced caspase3 down-regulation, it indicates inhibitory effect of ER66 on the ability of paclitaxel to induce apoptosis through caspase3 apoptosis pathway. To investigate the possible molecular mechanisms, we analyzed the expression and possible alterations of apoptosis-associated proteins including NF-KB/IK-Ba, JNK和AKT. The results reveal paclitaxel causes degradation of Iκ-Ba in BC-ER and BC-V cells. However, pretreatment with E2 attenuated the paclitaxel-induced IK-Ba down-regulation in BC-ER cells, although it has no effect in BC-V cells. Our studies have suggested that activation of the NF-κB/Iκ-Bαsignaling pathway maybe play an active role in mediating paclitaxel-induced apoptosis in BC-ER cells. We just preliminary studies JNK and AKT signaling pathway, the roles they play in ER66 mediating paclitaxel-induced apoptosis need further research.