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 (ER?), 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 ER? and have shown that ER? expression is associated with improved survival in TNBC patients. Additionally, the large majority of ER?+ 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 ER?+ TNBC, we have developed ER? expressing TNBC cell lines and have generated ER?+ and ER?- patient derived xenografts (PDX) from women with non-metastatic locally advanced TNBC. We show that activation of ER? 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 ER? in TNBC cells. Elevated cystatin levels lead to suppression of canonical TGF? 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 ER? 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 ER? will result in clinical benefit for patients with ER?+ TNBC by regulating a series of events that involves the induction of cystatins and suppression of canonical TGF? signaling. To test this hypothesis, the following Specific Aims are proposed: 1). Determine the role of cystatins, and their impact on TGF? signaling, in mediating the anti-cancer effects of ER? 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 ER?+ TNBC PDXs; 3). Elucidate the therapeutic efficacy of estradiol in ER?+ 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 ER? specific agonists for the treatment of ER?+ TNBC. Given the extremely poor outcomes in women with TNBC and the lack of targeted therapies for this group of patients, the proposed studies are of critical importance towards the goal of indentifying novel drug targets and therapeutic strategies to more effectively treat and manage this disease.