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Estrogen receptor isoforms ER-α and -β, although similar in structure and ability to bind 17β-estradiol (E2), have been shown to have opposite effects on cell growth and proliferation, with ER-α playing a stimulatory role and ERβ having an inhibitory function. Furthermore, differential expression of ER-α and -β has been reported in various cancer cell lines, including breast cancer cells, some of which have been shown to express one or both receptors (e.g., MCF-7, +ERα/+ERβ; T47D, +ERα/-ERβ; MB-MDA-231, -ERα/+ERβ). In this study, we investigated the expression levels of ERα, ERβ and ratio of ERβ/ERα in breast cancer cells with acquired resistance to the antiestrogens 4-hydroxytamoxifen (OHT, the active metabolite of tamoxifen) and fulvestrant (ICI 182,780, a selective estrogen receptor downregulator or SERD). Antiestrogen-resistant cells MCF7-T (tamoxifen-resistant) and MCF7-F (ICI-resistant) were previously established by growing hormone-sensitive MCF7 cells in hormone-free medium supplemented with OHT or ICI 182,780 (Fan et al., Cancer Res, 2006). In the present study, we examined, using Western blot analysis and commercially available ER isoform-specific antibodies, changes in protein levels of ERα and ERβ in MCF7-T and MCF7-F cells. In these cell lines, ERα levels changed dramatically, while ERβ levels remained unchanged, resulting in a high ratio of ERβ: ERα in MCF7-F cells and a low ERβ:ERα ratio in MCF7-T cells. To examine the receptor stability, MCF-7, MCF7-F and MCF7-T cells were treated with E2 (100nM), the phytoestrogen Apigenin (5μM), the SERD ICI 182,780 (100 nM), selective estrogen receptor modulators (SERMS; 1μM each of 4OHT, raloxifene or GW7604), or the histone deacetylase inhibitor TSA (300 nM) for 24 hrs. Protein levels of ERα and ERβ were detected by Western blot using specific antibodies. In all three cell lines, ERα was degraded by E2, Apigenin, ICI, GW7604 and TSA; however, the receptor was stabilized by OHT and raloxifene. ERβ was slightly degraded by E2 and TSA, but no effect of the other compounds on ERβ stability was observed. GST pulldown assays were used to show that ERα and ERβ can form a heterodimer in the presence of E2, ICI, or OHT, leading us to investigate whether ERβ can influence ERα transcription activity and sensitivity to antiestrogens. In an ERE-reporter gene assay, ERβ not only repressed E2-induced ERα transcription activity, but also reduced the sensitivity to inhibition of E2-induced ERα transcription activity by antiestrogens (ICI, 4OHT). The results of this study indicate that in response specific therapeutic agents, the differential stability of ERα and ERβ in breast cancer cell lines may change the ratio of these receptors and thus have an impact on breast cancer drug resistance.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA