P5-04-01: BP1, a Homeotic Transcription Factor, Indirectly Upregulates ER-alpha in ER Positive Cell Lines.

Abstract

Abstract Background: BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, including in 57% of ER positive and 89% of ER negative tumors. BP1 overexpression results in a more aggressive phenotype in breast cancer cells and larger tumors in women with breast cancer. We also observed the presence of mammary tumors in mice without estrogen supplementation after injection of MCF7 cells overexpressing BP1 compared with unsupplemented mice receiving MCF7 cells containing an empty vector. Our goal was to determine the pathway by which BP1 might cause this decrease in estrogen dependence of MCF7 cells. ER-alpha is a nuclear hormone receptor activated by estradiol and is a major target of anti-estrogen therapy. It is tightly regulated at the genomic and the non-genomic levels. Increased stability of ER-alpha is one mechanism by which cells can become ER-alpha independent. One way of altering ER-alpha stability is via p300 and BRCA1 since p300 acetylates and stabilizes ER-alpha, while BRCA1 destabilizes ER-alpha. We have previously shown that BP1 transcriptionally down-regulates BRCA1, suggesting that pBP1 may participate in this pathway. Materials and methods: Protein levels of BRCA1, p300, pS2, pBP1 and ER-alpha were determined by Western Blot analysis. RNA levels were measured by real-time PCR. A ChIP assay was performed to confirm binding of pBP1 to the EP300 gene and binding of ER-alpha to BP1. MCF7 parental cell lines were treated with 10nM E2 to induce the expression of pBP1. Experiments involving effects of siBP1 on p300 expression are underway. Results: Our data demonstrate that MCF7 ER+ cells engineered to overexpress BP1 exhibit increased ER-alpha protein, increased p300 and decreased BRCA1 protein as compared with control cells containing an empty vector and grown under the same conditions. Increased ER-alpha levels correlated with increased pS2 expression, a known downstream target of ER, demonstrating that there is more functional ER-alpha protein in cells overexpressing BP1. ChIP assays showed that pBP1 binds in the first intron of EP300-Histone acetyltransferase (p300). ChIP assays also showed that ER-alpha binds to an estrogen response element (ERE) upstream of the BP1 gene. MCF7 parental cell lines, when serum starved and treated with E2, showed increased BP1 expression compared with cells grown in charcoal stripped serum. Thus, BP1 may increase the stability of ER-alpha by decreasing BRCA1 and increasing p300. Conclusions: pBP1 binds and transcriptionally activates p300, leading to increased ER-alpha stability, while also partially repressing BRCA1. Moreover, ER-alpha, via a feed-forward mechanism, binds to the BP1 gene and increases its expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-04-01.