Long-term fulvestrant treatment results in irreversible loss of estrogen receptor alpha expression in MCF-7 human breast cancer cells

Abstract

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Fulvestrant (Faslodex®, ICI 182,780) is a steroidal pure antiestrogen. It has a high binding affinity to the estrogen receptor alpha (ERα), competitively inhibits the binding of estrogen to the ER, and blocks ER dimerization. Moreover, it causes ER degradation. Fulvestrant has no known agonist activity, and has been approved for the treatment of breast cancer which has progressed on prior antiestrogen therapy in postmenopausal women. Although this treatment will provide clinical benefit for women with advanced breast cancer, fulvestrant resistant disease will eventually occur. To understand the mechanism of fulvestrant resistance in order to evaluate further treatments, we developed a unique fulvestrant resistant model (MCF-7/ICI) using MCF-7 ERα positive human breast cancer cells by culturing the cells for twelve months in estrogen-free medium containing 1 μM fulvestrant to mimic postmenopausal conditions. MCF-7/ICI cells had become ERα negative as determined by real-time RT-PCR, Western blot analysis, and ERE-luciferase transfection assays. ERβ mRNA expression level did not change compared to the parental MCF-7 cells measured by real-time RT-PCR. Both parental MCF-7 and MCF-7/ICI cells were essentially ERβ negative as the mRNA ratio of ERβ:ERα was 1:7500. Interestingly, MCF-7/ICI cells remained ER negative after withdrawal of fulvestrant and switching the culture medium back to whole serum (estrogen con taining) medium for twelve months. Analysis of factors that may have been altered in the MCF-7/ICI cells showed that phosphorylation of p^44/42MAPK was significantly increased compared to parental cells. We also found that the MCF-7/ICI cells expressed significantly higher levels of epithelial growth factor (EGF) receptor mRNA and protein, as determined by real-time RT-PCR and Western blot analysis, respectively. However, EGFR overexpression was not due to gene amplification as indicated by FISH (Fluorescence in situ Hybridization) analysis. Consistent with EGFR overexpression, a specific EGFR tyrosine kinase inhibitor, Iressa (ZD1839), preferentially inhibits the growth of MCF-7/ICI cells relative to the parental MCF-7 cells in a dose dependent manner, which is consistent with a previous report (R.A. McClelland, et al. Endocrinology, 142:2776-88, 2001). In conclusion, MCF-7/ICI cells are the first MCF-7 subline to show irreversibly loss of ER expression after extended treatment of fulvestrant. The factors involving EGF receptor signaling transduction pathway are the potential targets for controlling the growth of MCF-7/ICI cells and may have clinical implications for further management of fulvestrant resistant ERα negative breast cancer. This work was supported by the Avon Foundation, the Lynn Sage Cancer Research Foundation, and the NIH SPORE in Breast Cancer CA 89018-03.