Activation of the transcription factor estrogen receptor α (ERα) and the subsequent regulation of estrogen-responsive genes play a crucial role in the development and progression of the majority of breast cancers. One gene target of ERα, growth regulation by estrogen in breast cancer 1 (GREB1), is associated with proliferation and regulation of ERα activity in estrogen-responsive breast cancer cells. The GREB1 gene encodes three distinct isoforms: GREB1a, GREB1b and GREB1c, whose molecular functions are largely unknown. Here, we investigate the role of these isoforms in regulation of ERα activity and proliferation. Interaction between GREB1 and ERα was mapped to the amino terminus shared by all GREB1 variants. Analysis of isoform-specific regulation of ERα activity suggests none of the GREB1 isoforms possess potent co-regulator activity. Exogenous expression of GREB1a resulted in elevated expression of some ER-target genes, independent of ERα activity. Despite this slight specificity of GREB1a for gene regulation, exogenous expression of either GREB1a or GREB1b resulted in decreased proliferation in both ER-positive and ER-negative breast carcinoma cell lines, demonstrating an ER-independent function of GREB1. Interestingly, we show an increase in the expression of GREB1b and GREB1c mRNA in malignant breast tissue compared to normal patient samples, suggesting a selective preference for these isoforms during malignant transformation. Together, these data suggest GREB1a has an isoform-specific function as a transcriptional regulator while all isoforms share an ER-independent activity that regulates proliferation.

Development of the mammary gland requires both proper hormone signaling and cross talk between the stroma and epithelium. While estrogen receptor (ERα) expression in the epithelium is essential for normal gland development, the role of this receptor in the stroma is less clear. Moreover, several lines of evidence suggest that mouse phenotypes of in utero exposure to endocrine disruption act through mesenchymal ERα in the developing fetus. We utilized a Twist2-cre mouse line to knock out mesenchymal ERα. Herein, we assessed mammary gland development in the context of mesenchymal ERα deletion. We also tested the effect of in utero bisphenol A (BPA) exposure to alter the tumor susceptibility in the mouse mammary tumor virus-neu (MMTV-neu) breast cancer mouse model. Mesenchymal ERα deletion resulted in altered reproductive tract development and atypical cytology associated with estrous cycling. The mammary gland demonstrated mature epithelial extension unlike complete ERα-knockout mice, but ductal extension was delayed and reduced compared to ERα-competent mice. Using the MMTV-Neu cancer susceptibility model, ERα-intact mice exposed to BPA had reduced tumor-free survival and overall survival compared to BPA-exposed mice having mesenchymal ERα deletion. This difference is specific for BPA exposure as vehicle-treated animals had no difference in tumor development between mice expressing and not expressing mesenchymal ERα. These data demonstrate that mesenchymal ERα expression is not required for ductal extension, nor does it influence cancer risk in this mouse model but does influence the cancer incidence associated with in utero BPA exposure.