A new regulator of mitochondrial OXPHOS in hepatocytes and HCC cell lines: the AAA plus ATPase RuvBL1

Hepatocellular carcinoma (HCC) is currently a major challenge in medicine for its poor prognosis and lack of effective therapeutic options. The AAA+ ATPase RuvBL1 associates with Hsp90 in several multiprotein complexes regulating key cellular pathways such as cell proliferation, gene expression, chromatin remodeling and telomere maintenance. In several human cancer, including HCC, RuvBL1 overexpression correlates with a poor prognosis. Despite the several functions potentially regulated by RuvBL1, its role in the onset and progression of HCC is still unknown. We had previously generated a RuvBL1 hepatocyte-specific knock-out mouse model and observed by TEM major structural alterations in mitochondria of hepatocytes. Moreover, by a 2D-proteomic screening, we found that several key mitochondrial proteins and Hsp90β were significantly reduced in RuvBL1-silenced hepatoma cells in vitro. Therefore, we aimed to dissect the role of RuvBL1 as a potential regulator of mitochondria functionality in HCC cells. First, we found that RuvBL1 targeting by RNAi significantly impairs mitochondrial respiration (OXPHOS) in normal hepatocytes (AML-12) and hepatoma cell lines (Hepa1-6, HepG2, Huh7, Hep3B). Live-cell imaging experiments showed a significant increase of mitochondrial ROS production and mitochondrial depolarization in RuvBL1-silenced cells. We next explored the RuvBL1-Hsp90 axis in mitochondrial functionality. We found that RuvBL1 is a positive regulator of HSP90AB1 promoter and mRNA levels, both in hepatoma and normal hepatocyte cell lines. Interestingly, Hsp90 inhibitors (17-AAG, VER-50589) effectively reduced mitochondrial OXPHOS paralleling RuvBL1 silencing. Analyzing the LIHC-TCGA database, we found a significant correlation between RUVBL1 and HSP90AB1 expression in HCC patients, consistently with our in vitro data. Finally, Gene Ontology analysis performed on genes differentially expressed in high- vs low-RUVBL1 HCC samples in the TGCA database, highlighted a significant enrichment of mitochondria-related terms. In conclusion, our data uncover a novel role of RuvBL1, possibly through its interaction with Hsp90β, in mitochondria integrity and function.