Role of BP1, a Novel Transcription Factor, in Antiestrogen Resistance.

Abstract

Abstract Background: Breast cancer patients with early stage, estrogen receptor (ER) positive tumors generally respond well to adjuvant treatment with the antiestrogen tamoxifen. The result is a 40-50% decrease in annual risk of recurrence. However, almost all patients with metastatic breast cancer and about half of the patients who receive tamoxifen exhibit recurrence due to resistance to antiestrogens. Through a better understanding of the mechanisms of resistance to antiestrogen therapy, it may be possible to design more effective treatment for those women with ER positive tumors who develop resistance, and possibly for those women with ER negative tumors, who do not respond to antiestrogen therapy. We are studying a homeotic transcription factor, BP1, that may be involved in antiestrogen resistance. BP1 is upregulated in 80% of invasive ductal breast tumors and is associated with aggressive tumors: 100% of ER negative tumors were BP1 positive, compared with 73% of ER positive tumors. We have evidence that BP1 protein (pBP1) is involved in both genomic and non-genomic ER signaling.Materials and Methods: MCF-7 derivatives overexpressing BP1, called O2 and O4 cells, were grown in serum free medium for 48 hr., followed by Western blot analysis to assess the level of phosphorylated Akt and phosphorylated ER. An electrophoretic mobility shift assay (EMSA) was performed with pBP1 transcribed and translated in wheat germ extract to demonstrate pBP1 binding to a specific DNA sequence. Cell lines were immunostained to determine levels of ER protein.Results: (1) Genomic signaling: There is a consensus DNA binding site in an intron of the ER. pBP1 binds to that site, shown by EMSA. Immunostaining of O2 and O4 cell lines overexpressing pBP1 demonstrates that there is reduced ER protein on cells overexpressing BP1, consistent with the idea that BP1 directly represses the ER and that BP1 is more frequently expressed in ER negative tumors than in ER positive tumors. (2) Non-genomic signaling: phosphorylation of Akt was examined in O2 cells after growth in phenol red-free, serum free medium. Using a phosphorylation-specific antibody, we observed phosphorylation of Akt in O2 cells. In contrast, there was little or no phosphorylation of Akt in empty vector control cells. Phosphorylation of the ER was assessed in O4 cells, where there was a detectable increase in ER phosphorylation in the cells overexpressing BP1 compared with empty vector controls.Discussion: Our data support the hypothesis that pBP1 binds to the ER, directly repressing its activity, which could lead to estrogen independence. Moreover, we have evidence that high levels of pBP1 lead to increased phosphorylation of both Akt and the ER, part of a non-genomic signaling pathway that causes estrogen independence. Moreover, in collaboration with Dr. Barbara Vonderhaar, we have shown that 20% of the tumors in mice injected with O2 or O4 cells are estrogen independent, consistent with these data. Thus, we hypothesize that BP1 is intimately involved in antiestrogen resistance, making it a strong potential therapeutic target. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5144.