Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation.

Altered lipid metabolism and lipid droplet (LD) dynamics are features of certain hepatocellular carcinoma (HCC) subtypes, but the molecular mechanisms governing LD trafficking and catabolism in HCC cells remain unclear. The small GTPase Rab5, a key regulator of early endosomal dynamics, has been previously observed to localize to the surface of LDs and participate in LD degradation through microlipophagy. However, the regulation of Rab5-LD interactions and its functional consequences in HCC cell metabolism and proliferation have not been elucidated. In this study, we explored the role of Rab5 in governing LD homeostasis and its impact on HCC cell proliferation. We found that GTP-bound Rab5 mutants (Q79L) exhibited increased association with LDs compared with the GDP-bound mutants (S34N) and WT Rab5. Acute nutrient starvation enhanced Rab5 GTP loading and recruitment to LDs, indicating that Rab5's GTPase cycle regulates its LD localization. Importantly, inhibition of Rab5 GTP binding led to increased LD accumulation, reduced mitochondrial respiration, and impaired proliferation in HCC cell lines. Transcriptomic analyses and tissue microarray immunohistochemistry further revealed that Rab5 is significantly overexpressed in HCC patient samples. These findings suggest that Rab5 GTP binding is an important contributor to LD dynamics in HCC cells, helping to govern LD turnover to sustain mitochondrial energy production and support cancer cell proliferation.