Estrogen Receptor A Mediates Breast Cancer Cell Resistance to Paclitaxel through Inhibition of Apoptotic Cell Death Meihua Sui, 1 Yi Huang, 2 Ben Ho Park, 2 Nancy E. Davidson, 2 and Weimin Fan 1 1 Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina and 2 The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland Abstract Estrogen receptors (ER) are expressed in f65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ERÀ) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfec- tion of ERA expression vectors into ERÀ breast cancer BCap37 cells. We showed that 17-B estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERA but has no influence on the ERÀ parental cells. Further analyses indicate that expression of ERA in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERA-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Addi- tionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERA and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ER- mediated resistance to paclitaxel and possibly other antineo- plastic agents. [Cancer Res 2007;67(11):5337–44] Introduction Estrogen receptors (ER) are transcriptional factors that play an important role in the development and progression of breast cancers (1–3). Cumulative analysis of tumor biopsies has shown that ERs are present in f65% of human breast tumors (4). It has long been known that breast cancers that express the ERa protein (ER+) behave in a fundamentally different fashion than ER-negative (ERÀ) tumors with regard to their response to hormonal therapies (4–6). In recent years, data from clinical trials or retrospective analyses suggested that ER status might also affect the efficacy of chemotherapy (7–10). Specifically, it has been observed that some chemotherapeutic agents may be less effective in patients with ER+ tumors than those with ERÀ tumors. These findings indicate that ER status may play an important role in determining the sensitivity of breast tumors to chemotherapy, although the mechanisms underlying ER-mediated drug resistance are not entirely clear (8, 11–13). Taxanes (paclitaxel and docetaxel), a novel class of naturally occurring antimicrotubule agents, represent active chemothera- peutic agents developed in the last two decades for the treatment of malignancies, including breast cancer (14, 15). However, not all breast tumors are sensitive to taxanes. Evidence is accumulating that improvements in taxane-based adjuvant chemotherapy dis- proportionately benefit patients with ERÀ breasttumors(9,16,17). Indeed, such a finding has also been observed in our in vitro experiments (18). In earlier studies, we characterized the possible correlation of the activation of nuclear factor-nB (NF-nB)/InB pathway with the susceptibility of tumor cells to paclitaxel-induced apoptosis (18–20). We found that the breast cancer cell line MCF-7 was highly resistant to paclitaxel-induced apoptosis although it was still responsive to paclitaxel in microtubule bundling and mitotic arrest (18). However, we did not appreciate that this resistance might be associated with ER status. Recently, we found that both paclitaxel-sensitive cell lines (human breast cancer cell line BCap37 and ovarian cancer cell line OV2008) used in this comparative study are ERÀ (see Fig. 1A ). This finding raised the possibility that the insensitivity of MCF-7 cell line to paclitaxel-induced apoptosis might be correlated to its ER. Therefore, in this study, we determined whether the differential sensitivity of breast cancer cells to paclitaxel might be a consequence of ERa expression. Through stable transfection of an ERa expression vector into ERÀ BCap37 cells, we found that, in the presence of 17-h estradiol, the expression of ERa in BCap37 cells is clearly associated with decreased sensitivity to paclitaxel. Analyses including DNA fragmentation, flow cytometry, and terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assays indicate that 17-h estradiol significantly interferes with the ability of paclitaxel to induce apoptosis in BCap37 cells transfected with ERa, but not in parental ERÀ BCap37 cells. Moreover, ICI 182,780 (Fulvestrant), a steroidal pure antiestrogen agent (21, 22), dramatically down-regulates ERa protein levels and nearly completely reverses the resistance to paclitaxel in BCap37 cells transfected with ERa. Meanwhile, the combination of ICI 182,780 was also found to sensitize ER+ MCF-7 and T47D cells to paclitaxel, which provided additional evidence that expression and subsequent activation of ERa are associated with resistance to paclitaxel in breast cancer cells. Materials and Methods Cell culture and agents. HumanbreastcancercelllinesBCap37(19,20), BCap37 transfected with pIRES-ERa expression vector (BC-ER), or empty vector (BC-V) were maintained in RPMI 1640, whereas MCF-7 and T47D cells were cultured in DMEM supplemented with 10% fetal bovine serum. Requests for reprints: Weimin Fan, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Charleston, SC 29425. Phone: 843-792-5108; Fax: 843-792-0368; E-mail: fanw@musc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-4582 www.aacrjournals.org 5337 Cancer Res 2007; 67: (11). 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