Understanding the role of estrogen receptor beta in triple negative breast cancers

Estrogen signaling is primarily mediated by two estrogen receptors (ERs), ERα and ERβ. ER is expressed in ∼70% of breast cancers and is an important diagnostic and therapeutic target. Approximately 10-15% of breast cancers lack expression of ERα, its target gene progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) and are known as triple negative breast cancers (TNBCs). Based on in vitro and clinical data, it is hypothesized that ERβ could be targeted with selective ligands to inhibit the growth of TNBCs. In order to identify and characterize subtype selective ligands in breast cancer cells, two isogenic reporter cell lines with inducible ERα or ERβ expression and a stably integrated estrogen responsive luciferase reporter were developed. These cell lines were highly sensitive to estrogenic ligands and could be used to characterize known subtype selective ligands. In order to assess the effects of ERβ expression and ligand treatment on the growth of TNBC cells in vitro and in vivo, the tumorigenic triple negative MDA-MB-468 cell line was engineered with inducible expression of full length ERβ. These cells were then used to assess growth effects and globally identify the ligand dependent and independent ERβ target genes using RNA sequencing. In order to specifically detect full length ERβ in ERα negative breast cancers, MDA-MB-468-ERβ xenograft tumor tissues were used to optimize immunohistochemical protocols for detecting full length ERβ in breast cancer tissues. The expression of full length ERβ was then confirmed in a subset of ERβ/PR/HER2 negative breast cancers using a cohort of triple negative breast cancers. This comprehensive study provides tools to identify subtype selective ligands and detect ERβ expression in clinical samples, confirms the expression of full length ERβ in a subset of TNBCs, demonstrates the growth inhibitory effects of ERβ expression and activation in vitro and in vivo, and globally identifies ERβ target genes in the context of triple negative breast cancer cells. The results ultimately provide a foundation on which to further develop ERβ selective ligands with the aim of inhibiting the growth of triple negative, ERβ-positive breast cancers.