PROJECT SUMMARY/ABSTRACT Triple Negative Breast Cancer (TNBC) affects approximately 20% of breast cancer (BC) patients and is the most aggressive sub-type of BC, in part due to the lack of therapeutic options available for patients. Our laboratory has demonstrated that Estrogen Receptor Beta (ERβ) protein is detectable at moderate to high levels in approximately 30% of TNBCs, and BC patients whose tumors retain ERβ expression have enhanced therapeutic response, lower rates of breast cancer recurrence, and improved disease-free survival. In addition, we have shown that activation of ERβ by estradiol (E2) decreases TNBC cell proliferation, invasion, and migration, demonstrating that ERβ functions as a tumor suppressor and is a relevant and novel drug target. To determine the mechanisms by which ERβ elicits its tumor suppressive effects, we have for the first time elucidated the ERβ transcriptome and cistrome in TNBC cells. Through pathway analysis of our gene expression studies, we identified the NFκB pathway as one of the most suppressed pathways in response to ligand-mediated activation of ERβ. Among the most down-regulated genes following E2 treatment were members of the interleukin (IL) family, which are known NFκB target genes. Chromatin Immunoprecipitation followed by sequencing (ChIPseq) for ERβ in TNBC cells revealed that ERβ was not only associated with EREs following E2 treatment, but was also enriched around NFκB binding sites. In fact, 12% of all ERβ binding sites encode NFκB response elements. Using an NFκB reporter construct and gene expression studies, we have shown that ligand-mediated activation of ERβ suppresses canonical NFκB signaling and blocks the activating functions of TNFα. We have also shown that this suppression is a result of chromatin modification. Suppression of NFκB signaling is known to inhibit proliferation and invasion of TNBC cells in vitro and decreased expression of NFκB target genes is associated with improved survival of TNBC patients, yet direct suppression of NFκB signaling has toxic side effects. However, we expect indirect or cell-specific blockade of the NFκB pathway in TNBC patients would suppress tumor progression and improve outcomes. In light of these observations and the known oncogenic properties of NFκB in TNBC, this proposal aims to address the central hypothesis that ERβ elicits its tumor suppressive effects in TNBC by suppressing the NFκB signaling pathway. In Specific Aim 1, we will determine the mechanisms by which ERβ suppresses NFκB signaling in TNBC using multiple cell line model systems. In Specific Aim 2, we will utilize a novel ERβ transgenic mouse model to determine the impact of ERβ expression and activation on TNBC progression and assess alterations in NFκB signaling during this process. Additionally, we will employ patient-derived organoids to evaluate the efficacy of ERβ targeted agents and its impact on NFκB signaling. Completion of these studies will define the molecular mechanism...