Estrogen-Estrogen Receptor axis in non-transformed breast epithelial cells: studies beyond MCF-7



 DESCRIPTION (provided by applicant): Estrogen (E2) acting through the nuclear receptor estrogen receptor alpha (ERα) plays a major role in breast cancer. Approximately 70% of breast cancers express ERα and patients with ERα-positive breast cancers receive anti-estrogens. However, resistance to anti-estrogen therapy is a major clinical problem. There are at least two critical unresolved issues in this field: 1) in the normal breast, ERα is expressed in 10-20% of non- dividing cells. These ERα-positive cells support proliferation of ERα-negative cells in response to E2 through paracrine action. In contrast to normal cells, ERα-positive cancer cells themselves proliferate in response to E2. Mechanisms governing switch in this ERα function are largely unknown and is the focus of this proposal; 2) Mechanisms of anti-estrogen resistance. Most of our current knowledge of ERα biology including genome- wide binding pattern (ERα cistrome), the role of pioneer factors/chromatin modifications in guiding ERα cistrome, and E2-regulated gene expression pattern (E2-transcriptome) are from studies using the cancer cell line MCF-7 but not normal/non-transformed cells because it has not been technically possible to culture ERα- expressing non-transformed cells. This limitation has prevented us from understanding the function of ERα in the normal breast and mechanisms that govern cancer-associated shift in ERα function. To address these critical issues, we generated several ERα-positive breast epithelial cell lines by propagating cells from normal breast using recently described epithelial cell-reprogramming assay followed by immortalization using the hTERT. Consistent with the predicted expression pattern of ERα in primary cells, ERα is expressed in the luminal progenitor and mature cell but not in stem cell fraction of these cell lines, which authenticates the relevance of the system. RNA-seq analysis of one of these cell lines showed dominant action of ERα:E2 in repressing gene expression. In contrast, ERα-signaling involved both activation and repression in cancer cells and was well integrated with PI3K-Cyclin D1-cMyc proliferation network as well as an atypical ubiquitination system not seen in normal cells. Using these new resources, we will address the hypothesis that switch in ERα cistrome and E2-transciptome from a repressor mode to a activator mode is a critical early step in ERα- positive breast cancer. The specific aims are to: 1) Define ERα cistrome that is unique to non-transformed breast epithelial cells; 2) Investigate signaling nodes that govern ERα function in non-transformed and transformed breast epithelial cells by integrating ERα cistrome with transcriptome. Impact: To our knowledge, this is the first study to determine ERα function in non-transformed breast epithelial cells. The resources developed will be valuable to the research community to reassess signaling networks associated with ERα-positive breast cancer and anti-estrogen resistance. In particular, signaling defects linked to genomic aberrations uniquely enriched in ERα-positive breast cancers can be investigated and drugs targeting specific aberration can be identified using this resource.