| The estrogen receptor (ER) plays a major role in breast cancer progression, and ER+ tumors respond favorably to hormonal manipulation. The selective estrogen receptor modulator (SERM) tamoxifen (Tam) induces remissions in most ER+ patients. However, acquired resistance is often observed. Tam-resistant breast cancer is sensitive to other antiestrogenic compounds, but resistance to these agents has also been described, illustrating a major limitation to antiestrogen therapy. Therefore, we investigated a ligand-independent approach for treating Tam-resistant breast cancer by targeting the molecular chaperone Hsp90.; The ER exists in a multi-protein complex containing Hsp90, which regulates the activity and stability of the receptor. Hsp90 regulates the stability of other proteins relevant to breast cancer, including Akt and Raf-1. The benzoquinone ansamycin antibiotic geldanamycin (GA) and its clinically relevant analog, 17-demethoxy-17-allylaminogeldanamycin (17AAG), bind to Hsp90 and induce the degradation of Hsp90 clients. In these studies, we show that GA depletes ER levels in Tam-resistant cell lines, and the Hsp90 clients Akt and Raf-1. Unexpectedly, Tam inhibited GA-induced degradation of the ER, but not Akt and Raf-1. This effect was consistent in vivo, where ER levels were measured in tumor xenografts growing in Tam-supplemented mice. However, Tam-stimulated tumor growth was inhibited by 17AAG, and tumor Akt and Raf-1 levels were downregulated. Immunoprecipitation experiments showed that Tam does not inhibit GA-induced changes in the ER-chaperone complex, suggesting an alternate mechanism for the inhibition of GA-mediatied ER degradation. Through cell fractionation, immunostaining, and chromatin immunoprecipitation experiments, we have found that the mechanism involves prolonged association of the ER with the DNA in the presence of Tam, which leads to nuclear accumulation of the ER and sequestration from the proteasome. Furthermore, inhibition of GA-induced ER degradation was inhibited by another SERM, Raloxifene, indicating that the effect is not Tam-specific.; Based on its ability to downregulate critical signaling proteins involved in breast cancer, including the ER, 17AAG may provide a useful alternative for patients that have failed hormonal therapy. Because SERMs inhibit the degradation of ER protein induced by GA, they may compromise the efficacy of GA on ER activity, and combined therapy should be approached with caution. |
