We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha (ERalpha)However, the relevance of ERbeta in mediating endoxifen action has yet to be explored. Here, we characterize the molecular actions of endoxifen in breast cancer cells expressing ERbeta and examine its effectiveness as an anti-estrogenic agent in these cell lines.

MCF7, Hs578T and U2OS cells were stably transfected with full-length ERbeta. ERbeta protein stability, dimer formation with ERalpha and expression of known ER target genes were characterized following endoxifen exposure. The ability of various endoxifen concentrations to block estrogen-induced proliferation of MCF7 parental and ERbeta-expressing cells was determined. The global gene expression profiles of these two cell lines was monitored following estrogen and endoxifen exposure and biological pathway analysis of these data sets was conducted to identify altered cellular processes.

Our data demonstrate that endoxifen stabilizes ERbeta protein, unlike its targeted degradation of ERalpha, and induces ERalpha/ERbeta heterodimerization in a concentration dependent manner. Endoxifen is also shown to be a more potent inhibitor of estrogen target genes when ERbeta is expressed. Additionally, low concentrations of endoxifen observed in tamoxifen treated patients with deficient CYP2D6 activity (20-40 nM) markedly inhibit estrogen-induced cell proliferation rates in the presence of ERbeta, whereas much higher endoxifen concentrations are needed when ERbeta is absent. Microarray analyses reveal substantial differences in the global gene expression profiles induced by endoxifen at low concentrations (40nM) when comparing MCF7 cells which express ERbeta to those that do not. These profiles implicate pathways related to cell proliferation and apoptosis in mediating endoxifen effectiveness at these lower concentrations.

Taken together, these data demonstrate that the presence of ERbeta enhances the sensitivity of breast cancer cells to the anti-estrogenic effects of endoxifen likely through the molecular actions of ERalpha/beta heterodimers. These findings underscore the need to further elucidate the role of ERbeta in the biology and treatment of breast cancer and suggest that the importance of pharmacologic variation in endoxifen concentrations may differ according to ERbeta expression.

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