PROJECT SUMMARY/ABSTRACT Renal cell carcinoma (RCC) accounts for more than 430,000 cases of cancer worldwide and about 180,000 cancer-related deaths each year. Clear cell RCC (ccRCC) is the most frequent (75%–80%) subtype of RCC. Traditional chemotherapy and radiation therapy are largely ineffective in the treatment of all RCC subtypes. The development of multi-targeted tyrosine kinase inhibitors (TKIs) and immunotherapeutic agents notably changed the treatment paradigm of advanced kidney cancer. However, despite the therapeutic progress, complete and durable responses have been noted in only a few cases. Furthermore, responses to the available therapeutic agents remain unpredictable in individual ccRCC patients. We have shown previously that LDL cholesterol compromises the antitumor efficacy of clinically relevant TKIs. Our recent findings show that major lipoproteins (i.e. LDL, HDL, and VLDL) are equally effective in supporting growth of ccRCC cells and counteracting the antitumor activity of TKIs. In contrast to normal cells, ccRCC cells largely depend on exogenous cholesterol supply. The intracellular trafficking of exogenous lipoprotein-derived cholesterol appears to be distinct from movement of endogenously synthesized cholesterol. De novo synthetized cholesterol is transported from the endoplasmic reticulum directly to the plasma membrane and to the acyl-CoA:cholesterol acyltransferase whereas lipoprotein-derived cholesterol is distributed through the NPC1-dependent endosomal trafficking system. NPC1 plays a critical role in maintaining adequate cholesterol supply in cells that cannot produce endogenous cholesterol. Expression of NPC1 is increased in ccRCC at mRNA and protein levels, and high expression of NPC1 is associated with poor prognosis based on TCGA data analysis. Importantly, trafficking of de novo synthetized cholesterol in normal cells is not affected by pharmacological or genetic inhibition of NPC1, in contrast to trafficking of exogenously-derived cholesterol. Our recent findings support this observation showing that ccRCC cells are particularly sensitive to NPC1 inhibition. Given that ccRCC cells have redundant mechanisms of cholesterol acquisition, we hypothesize that only concomitant targeting of all sources of cholesterol acquisition or common routes of intracellular cholesterol trafficking would deprive tumor cells of cholesterol supply. To test our hypothesis and to elucidate the role of NPC1 as a novel therapeutic target and a potential prognostic biomarker in ccRCC, we propose the following Specific Aims: (1) Evaluate NPC1 as a potential therapeutic target in ccRCC; (2) Determine if expression of lipid biomarkers correlates with clinical outcomes in ccRCC.