Project Summary Triple Negative Breast Cancer (TNBC) is an aggressive subtype representing 15-20% of newly diagnosed breast cancer cases. Due to a lack of known molecular targets, treatment options for TNBC are limited, and 30-50% of patients acquire resistance to standard care. I aim to better understand the metabolic dependencies of TNBC cells to reveal novel targets that could be exploited therapeutically. TNBC cells tend to exist in a mesenchymal-like state, having undergone epithelial-to-mesenchymal transition (EMT) during which cells acquire a more motile and invasive phenotype. Because EMT is associated with poor breast cancer prognosis, understanding the molecular drivers and adaptations during this process will identify targetable dependencies of metastatic cancer cells. microRNA-200c (miR-200c) is well-established as a suppressor of EMT that tends to be lost in TNBC. Using miR-200c as a tool to push TNBC cells into a more epithelial-like phenotype, I have identified novel changes to cholesterol metabolism occurring during EMT. This project delineates how EMT regulates cholesterol metabolism and the functional consequences of these changes. In the F99 portion of this grant, I establish how reversal of EMT by miR-200c alters cholesterol metabolism proteins including the low-density-lipoprotein (LDL) receptor (LDLR) and Niemann-Pick-Type-C1 (NPC1). LDLR is responsible for endocytosing LDL particles, which are the primary source of cholesterol for breast tumor cells. After fusion of endocytic vesicles with the lysosome, NPC1 is specifically required for the processing and transport of cholesterol from lysosomes to other cellular components. These changes suggest an altered demand for cholesterol mesenchymal-like versus epithelial-like TNBC, the consequences of which are evaluated in Aim 1B of this project. My preliminary data also demonstrates that NPC1 activity is critical to survival of TNBC cells in vitro. In Aim 1B, I outline experiments to understand overall cholesterol uptake and intracellular cholesterol levels during EMT and delineate the critical role of NPC1 on TNBC cell viability. This work is the first to establish altered dependency and expression of LDLR and NPC1 in EMT and breast cancer progression. In the K00 phase of the proposed project, I will expand on my interest in the metabolic requirements of the metastatic cascade. I will move into investigating how extracellular mechanical cues driven by the extracellular matrix (ECM) drive metabolic adaptations to promote invasion and migration during metastasis. Further, I will evaluate whether the EMT phenotype of cells influences these adaptations. Gaining a better understanding of how cancer cell metabolism supports changing ECM and how mechanical demands encountered during metastasis will provide insights into development of therapeutic targets within tumor metabolism.