Project Summary/Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer and fourth leading cause of cancer deaths worldwide. The incidence of HCC is rising as key risk factors including obesity and heavy alcohol consumption are accelerating at rates greater than ever before. These risk factors drive hepatic steatosis and steatohepatitis, marked by the extreme accumulation of lipid droplets (LDs) in hepatocytes, leading to cirrhosis and HCC. While lipid synthesis a well-described phenotype in HCC, the role of LDs in propagating tumor formation and growth is poorly understood. Preliminary data from our laboratory suggests that the small GTPase Rab5 localizes to LDs and plays a critical role in trafficking to the lysosome, where LDs are catabolized by lysosomal acid lipase (LAL). Moreover, both Rab5 and LAL expression are increased in HCC tumors, consistent with cell biological studies showing elevated lipophagy in HCC cell lines vs normal hepatocytes. These data support the hypothesis that Rab5-LD interaction drives HCC tumor growth via lysosomal catabolism. In Aim 1, we will determine the mechanisms of Rab5 binding to LDs in HCC, and how this interaction impacts cancer cell energetics and proliferation. In Aim 2, we will determine the impact of Rab5-mediated lipophagy on HCC tumor growth in vivo using a sleeping beauty transposon transposase (SBTT) mouse model in which oncogenes are delivered to hepatocytes via hydrodynamic tail vein injection (HTVI). The results gained from the proposed research will provide a mechanistic understanding of lipid droplet trafficking and catabolism in hepatocellular carcinoma progression. Moreover, this research will aid in future development of larger research project grant applications.