Targeting RuvBL1 disrupts mitochondrial metabolism and structure in hepatocellular carcinoma.

[BACKGROUND & AIMS] The AAA+ ATPase RuvBL1 takes part in several biological processes, including chromatin remodelling and DNA repair, ribosome biogenesis, mTOR signalling, and oncogenic transformation. RUVBL1 overexpression correlates with poor survival in hepatocellular carcinoma patients. We previously found that RuvBL1 is a key regulator of liver glucose metabolism in mice. Here, we aimed at disentangling the metabolic function of RuvBL1 in HCC cells.

[METHODS] Non-transformed AML-12, primary mouse hepatocytes, HCC cell lines, and RuvBL1 mice were used (n=3). RuvBL1 was targeted by RNAi and by inhibition with CB-6644. Metabolomic profiling and mitochondrial functions were assessed by targeted GC/MS, Seahorse analysis, and ATP synthase activity. Mitochondrial morphology and membrane potential were investigated by fluorescence microscopy, High-Content Imaging, and TEM. Mitochondrial RuvBL1 was detected by WB, super-resolution microscopy, TEM, and PLA. Human HCC and normal liver samples from TCGA and GTEx databases were used for in silico analysis (T=369, N=160).

[RESULTS] Targeting RuvBL1 impairs mitochondria-centred metabolic processes, including amino acid metabolism, TCA cycle, and OXPHOS. Inhibition of RuvBL1/2 activity induces loss of cristae integrity, mitochondrial hyperpolarization and fragmentation, a phenotype paralleled by the hepatocytes of RuvBL1 mice. We detected RuvBL1 in proximity to mitochondrial ATP synthase, a previously unreported localization for this protein. Mechanistically, CB-6644 reduces ATP synthase-RuvBL1 interaction and impairs complex V activity even under a fuelled TCA cycle. In human HCC, higher RUVBL1 expression correlates with gene signatures associated with mitochondrial oxidative phosphorylation (FDR=5.64e), ATP synthase complex (FDR=6.03e), and poorer outcome (p=2e).

[CONCLUSIONS] Targeting RuvBL1 impairs complex V activity, disrupting mitochondrial metabolic functions and structural integrity. The mitochondrial functions of RuvBL1 may inform novel therapeutic strategies in the fight against hepatocellular carcinoma.

[IMPACT AND IMPLICATIONS] Metabolic reprogramming is a key feature driving HCC onset, progression, and plasticity, contributing to treatment resistance and poor prognosis. RUVBL1 overexpression correlates with reduced survival of HCC patients and has emerged as a potential metabolic modulator. In this study, we found that targeting RuvBL1 impairs its interaction with mitochondrial ATP synthase, disrupting mitochondrial metabolism and cristae structure. In human HCC samples, RUVBL1 expression correlates with hallmark mitochondrial metabolic processes. These findings may inform the development of targeted therapeutic approaches aimed at impairing the metabolic rewiring and plasticity of HCC.