PROJECT SUMMARY/ABSTRACT Breast cancer is the most diagnosed cancer type and the second leading cause of cancer-related death in women in the United States. For the receptor positive (RP) subtype comprising the majority of diagnoses, clinical interventions have been largely effective in limiting associated mortality when compared with other cancers. However, the remaining 20%, which comprise the triple-negative breast cancer (TNBC) subtype, lack known therapeutic targets and are the most clinically challenging. Cancers display altered metabolism upon carcinogenesis. However, the efficacy of targeting metabolism in cancer depends upon understanding metabolic dysregulation in the context of a specific oncogene. Our lab and others have shown that levels of c-MYC (MYC), a proto-oncogene that dynamically regulates numerous cellular functions during transformation, are increased in a majority of TNBC. MYC is known to regulate glucose and glutamine metabolism in cancer, but our lab has shown that MYC regulates another important bioenergetic pathway, fatty acid oxidation (FAO), in TNBC. Models for MYC-overexpressing TNBC display decreased bioenergetic metabolism and primary tumor growth upon inhibition of FAO, indicating novel reliance on that pathway. Here, we propose to investigate how TNBC permit increased FAO. Increased FAO necessitates alterations to fatty acid (FA) availability, and one fatty acid trafficking component, fatty acid binding protein 5 (FABP5), is upregulated in TNBC in a MYC-dependent manner. FABPs are lipid chaperones that bind cytosolic FA and other molecules, and can access a range of cellular compartments. Our data indicate that FABP5 loss in MO-TNBC is sufficient to cause defects in cell metabolism and proliferation. FABP5 may contribute to altered FAO and proliferation by facilitating increased FA supply at the mitochondria to permit increased oxidation. We hypothesize that increased trafficking of FA to the mitochondria by FABP5 facilitates altered FAO in a MYC-dependent manner. Accordingly, we propose investigation of the cellular biology and biochemistry of FA trafficking, FABP5 regulation and FAO in TNBC. Our strategy combines: utilization of high-content microscopy to visualize FA trafficking in vitro, carbon-tracing studies to follow FA metabolism in vivo and in vitro, mass spectrometry-based metabolomic analyses, pharmacological and genetic perturbations of FA trafficking, and conditional and constitutive MO-TNBC cell lines and tumor models. We expect that our investigation of mechanisms of FAO in MO-TNBC will advance understanding of how FA metabolism is regulated in cancer, and may identify novel therapeutic targets for the treatment of this clinically challenging breast cancer subtype.