Women with estrogen receptor (ER)-positive breast cancer are treated with hormonal agents targeting the ER, and can remain free of disease for many years. However, resistance to treatment eventually develops, and these women will thus suffer a recurrence of their cancer. In our current grant period, we explored our proposal that the key to the successful treatment of these women is to focus on identifying novel mechanisms of resistance, using metastatic tumors resistant to hormone therapy in which tumor evolution and clonal selection for resistance have already occurred. Using this innovative approach, we have already discovered several new individual resistance mechanisms, including reduced levels of two key negative regulators of resistance - Rho guanine dissociation inhibitor alpha (Rho GDIa) and the growth factor receptor scaffolding protein SLC9A3R1, both of which enhance activation of ERa via its phosphorylation. We will focus on these two resistance mechanisms, and determine their effect on cells with wild-type ERa as well as two specific ERa mutations (lysine 303 and tyrosine 537) that we were the first to discover in breast tumors, and which significantly affect hormone response. We will exploit these resistance mechanisms to exert more precise control over resistant tumor growth, combining hormonal and targeted therapy along with knowledge of the activation status of the key ERa growth pathway. Therefore, we now propose to validate specific resistance mechanisms associated with losses of Rho GDIa or SLC9A3R1 and their effects on ERa, to target these specific mechanisms, and to translate these findings to pinpoint predictive clinical biomarkers and potential treatment targets. Our Aims are: (1) To target SLC9A3R1 and Rho GDIa resistance mechanisms to restore hormone sensitivity and response to targeted therapies; (2) To determine how SLC9A3R1 and Rho GDIa.resistance pathways pact on mutant ERa function; and (3) To validate selected candidates from these two pathways for their ability to predict resistance to targeted hormone and biologic therapies, using our extensive retrospective clinical breast tumor resources. This work should lead to extended effective suppression of recurrence for the large numbers of women with ER-positive breast cancer who now daily fear reappearance of their disease in spite of continued endocrine treatment.