Abstract
BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Although mitochondrial metabolism contributes to tumorigenesis, the specific roles of individual mitochondrial components remain unclear.NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), a key subunit of mitochondrial complex I, has been implicated in non-hepatic malignancies, but its functional relevance in HCC is unknown. METHODS: We assessed NDUFS8 expression in HCC tissues and cell lines using TCGA datasets and patient specimens. Functional analyses-including mitochondrial assays, apoptosis, proliferation, and migration-were performed in NDUFS8-silenced, knockout, and overexpressing HCC cells. In vivo tumor growth was evaluated using xenograft mouse models. Mechanistically, mass spectrometry and immunoprecipitation identified HUWE1 as an E3 ligase responsible for NDUFS8 ubiquitination. RESULTS: NDUFS8 was significantly overexpressed in HCC tissues and cell lines, correlating with poor patient prognosis. NDUFS8 localized to mitochondria and promoted complex I activity and ATP production. Knockdown or knockout of NDUFS8 impaired mitochondrial function, increased ROS, disrupted redox homeostasis, induced apoptosis, and suppressed proliferation and migration of HCC cells. In contrast, NDUFS8 overexpression enhanced oncogenic behaviors. In vivo, NDUFS8 silencing via AAV delivery significantly inhibited xenograft growth and triggered apoptosis. Mechanistically, HUWE1 was identified as a specific E3 ligase that ubiquitinates NDUFS8 at lysine 88, regulating its stability. CONCLUSIONS: NDUFS8 is a mitochondrial regulator that promotes HCC progression through metabolic activation and is post-translationally modified by HUWE1. Targeting NDUFS8 or its regulatory axis may represent a promising therapeutic strategy for HCC.