PROJECT SUMMARY/ABSTRACT: In 2015 it is estimated that more than 1.6 million women will develop breast cancer world-wide, of which nearly 20% will be diagnosed with a form of the disease referred to as triple negative breast cancer (TNBC) that lacks expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2). For these reasons, targeted therapies are currently limited for this patient population. While chemotherapy improves the survival of TNBC patients, 5 year mortality rates approach 50% for individuals with residual disease after neoadjuvant treatment. It is therefore paramount to identify new drug targets and therapeutic strategies for women with TNBC. We and others have recently demonstrated that 30% of TNBCs express ERβ and have shown that ERβ expression is associated with improved survival in TNBC patients. Additionally, the large majority of ERβ+ TNBCs do not express the androgen receptor allowing for the potential expansion of endocrine related therapies into an additional subset of TNBC patients. To identify therapies that may have utility for the treatment of ERβ+ TNBC, we have developed ERβ expressing TNBC cell lines and have generated ERβ+ and ERβ- patient derived xenografts (PDX) from women with non-metastatic locally advanced TNBC. We show that activation of ERβ with estradiol significantly reduces cell proliferation and tumor growth, effects that appear to be mediated in part by a family of proteins known as the cystatins which are highly induced by ERβ in TNBC cells. Elevated cystatin levels lead to suppression of canonical TGFβ signaling, a pathway known to drive tumor progression, metastasis, and resistance to chemotherapy regimens. We have also shown that these same anti-cancer effects can be achieved with drugs such as LY500307 that specifically target ERβ allowing for the opportunity to develop cancer therapies that could avoid the negative side effects associated with estradiol. Based on these preliminary data, our central hypothesis is that therapeutic activation of ERβ will result in clinical benefit for patients with ERβ+ TNBC by regulating a series of events that involves the induction of cystatins and suppression of canonical TGFβ signaling. To test this hypothesis, the following Specific Aims are proposed: 1). Determine the role of cystatins, and their impact on TGFβ signaling, in mediating the anti-cancer effects of ERβ in TNBC and characterize the expression of these biomarkers in a large cohort of TNBC patients; 2). Characterize the in vivo effects of estradiol and LY500307 on ERβ+ TNBC PDXs; 3). Elucidate the therapeutic efficacy of estradiol in ERβ+ TNBC. To address these Specific Aims, we will employ multiple in vitro and in vivo approaches to fully characterize the mechanisms of action and anti-tumor activity of estradiol and ERβ specific agonists for the treatment of ERβ+ TNBC. Given the extremely poor outcomes in women with TNBC and the lack of targete...