Project Summary Breast cancer is the most commonly diagnosed cancer in women, with estrogen receptor positive (ER+) breast cancers accounting for 75% of cases. Endocrine therapies directed at blocking ER action are highly effective; however, 40% of women with ER+ tumors develop resistance and progress to metastatic disease. ER+ tumors relapse late, and tumor cells can remain quiescent for years to decades. Progress in the treatment of metastatic breast cancer is limited by strategies that primarily target rapidly proliferating tumor cells. Contributing factors to advanced disease progression include breast cancer stem cells (CSC), which are poorly proliferative and exist as minority populations in therapy resistant tumors. We identified SRC-3 (steroid receptor [SR] co-activator 3) as a novel cytoplasmic binding partner of PELP1. Similar to SRC-3, PELP1 is an ER co-activator, and dynamically shuttles between the nucleus and cytoplasm to act as a nuclear co-activator and cytoplasmic scaffolding protein for growth factor and steroid receptors. PELP1 is primarily nuclear in normal breast, but increased cytoplasmic localization of PELP1 is an oncogenic event that promotes disease progression by unknown mechanisms. We showed PELP1/SRC-3 cytoplasmic complexes drive breast CSC phenotypes and genes associated with pro-survival in ER+ breast cancer models. SRC-3 inhibition disrupts complex formation and cytoplasmic PELP1-induced tumorspheres. Top candidates identified from RNA-seq analysis include PFKFB family members, which are bi-functional kinase/phosphatases that have roles in cancer metabolism and CSC biology. PFKFB3/-4 co-purified with PELP1/SRC-3 complexes; inhibition of PFKFB3/-4 blocked PELP1/SRC-3 complex formation and biology. Remarkably, PELP1/SRC-3 CSC biology is phenocopied in tamoxifen-resistant (TamR) and paclitaxel-resistant (TaxR) models. Herein, we hypothesize that PELP1/SRC-3 complexes amplify signaling inputs to PFKFB family members that mediate altered metabolic pathways required for resistant ER+ tumor cell populations. We will: 1) identify signaling pathways essential for PELP1/SRC-3 driven therapy resistance using mass cytometry, and 2) determine the therapeutic benefits of targeting PELP1/SRC-3/PFKFB complexes in vivo to block cancer progression and metastasis. Our long-term objectives are to identify non-ER therapeutic targets that can be developed as combination strategies to eliminate therapy resistant tumor cells in ER+ breast cancer. During the K22 award, we expect to define the molecular links between cancer cell metabolism and oncogenic events in breast cancer progression, metastasis, and examine the benefits of targeting this pathway to impair late recurrence. This proposal will provide a solid foundation for the candidate’s goal of moving towards translational cancer research during her transition to independence. Delineating the key players will fundamentally redefine standard care options to target therapy-resistant pop...