Background: The estrogen receptor (ER) is expressed in ≈70% of sporadic breast cancer and activates genes driving cell proliferation and tumorigenesis. We have previously performed genome-wide analysis of ER binding sites in MCF-7 breast cancer cells and identified distinct mechanisms of tumorigenic ER signaling. Using EpCAM and CD49f as markers to enrich for ER-positive (ER+) cells obtained from primary non-malignant breast tissue, we seek to elucidate differences in ER signaling between normal and primary and metastatic ER+ breast cancer cells.

Methods: Viable breast epithelial cells were obtained from patients undergoing reduction mammoplasties, and ER+ breast cancer cells from primary tumors and metastatic effusions. Following dissociation into single cells, EpCAM+ cell subpopulations were isolated and stimulated with estradiol. Gene expression microarray analysis, chromatin immunoprecipitation and DNA sequencing (ChIP-seq) on transcription factors and histone modifications as well as DNAse I hypersensitivity assays (DHS) were performed, and compared to MCF-7 breast cancer cells.

Results: Triplicates of normal, ER+ breast cancers, and metastatic ER+ cancer were analyzed. Gene expression profiles revealed differences in estradiol regulated genes between primary normal, breast tumor, and metastatic ER+ breast cancer cells. Genes that promote cell cycling and cell proliferation were downregulated in non-malignant tissue but were upregulated in breast cancer cells. Our ChIP-seq results showed differential binding of ER between normal and ER+ breast cancer with little common overlap, and motif analysis of these binding sites demonstrated the enrichment of ERE motifs in common sites, TCF12 motifs in unique normal sites, and FOXA1 motifs in unique breast cancer sites. Analyses of the distribution of histone modifications and DHS regions demonstrated distinct patterns at shared, normal, and breast cancer ER binding sites, suggesting functionality and further validating differential ER binding.

Conclusions: There are contrasting differences in ER signaling between normal mammary and ER+ breast cancer cells, with estrogen appearing to have anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in BC. ER ChIP-seq has identified unique motifs, distribution of histone modifications, and DHS regions specific to unique normal, cancer and shared ER binding sites. Our studies point to TCF12 as a potential ER pioneer cofactor in non-malignant breast tissue and provide more data in support of FOXA1 as an important ER coregulator in ER+ breast cancer. Our data provides evidence for key alterations in ER-signaling during tumorigenesis and could lead to the identification of novel strategies to target breast cancer specific ER signaling.

Citation Format: David Chi, Housheng He, Tony Yeung, Rinath Jeselsohn, Stuart Schnitt, Judy Garber, Andrea Richardson, Elgene Lim, Myles Brown. Differences in estrogen receptor signaling in normal mammary epithelial cells and ER-positive primary breast tumors and metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2013-2313