Hepatocellular carcinoma (HCC), the predominant primary cancer of the liver, is 4th leading cause of cancer deaths, and the fastest growing malignancy in the US. Advanced-stage HCC remains largely incurable due to a dismal response rate (<20%) and therapeutic resistance. The current obesity epidemic has been associated with the rising prevalence of metabolic (dysfunction) associated fatty liver disease (MAFLD) and its inflammatory component, non-alcoholic steatohepatitis (NASH), which can lead to HCC. MAFLD-HCC incidence is increasing dramatically, underscoring an unmet medical need for new diagnostic and treatment strategies. Here, we describe a novel tumor suppressor role of the Hypoxia-Associated Factor, HAF, in MAFLD-HCC. Both global haploinsufficiency and hepatocyte-specific deletion of HAF in mice result in HCC with hallmarks of NASH including severe steatosis with hepatocyte degeneration (hepatocyte ballooning), fibrosis, and increased inflammatory cell infiltration. HAF loss in both mouse models or by siRNA transfection in HCC cells is associated with decreased activation of the p65/p50 NF-κB transcriptional subunits, and in decreased levels of their upstream regulators, TAK1 and NEMO. Endogenous HAF forms a complex with NEMO and TAK1, suggesting that HAF modulates the NF-κB pathway by directly modulating the stability of these proteins, potentially through HAF’s E3 ubiquitin ligase activity. HAF knockdown was associated with increased spontaneous apoptosis, whereas HAF overexpression protected cells against TNF-induced cell death, suggesting that HAF may play a tumor suppressor role by protecting cells against death associated with liver inflammation (hepatitis) that can lead to HCC. Indeed, HAF levels are suppressed by conditions prevalent during hepatitis such as hypoxia and elevated TNF or TGF-β, whereas HAF are increased by DNA damage, suggesting that HAF may contribute to NF-κB activation in response to genomic instability in pre-neoplastic hepatocytes. Significantly, HAF was highly expressed in most cases of human hepatitis but was undetectable in 94% of human HCCs examined (65 cases). Thus, our hypothesis is that HAF plays a novel tumor suppressor role in HCC by facilitating NF-κB activation that promotes the survival of hepatocytes during hepatitis. Suppression of HAF by hypoxia or inflammatory cytokines during hepatitis results in increased cellular turnover that drives progression to NASH and HCC. Our overall goal is to identify new predictive/prognostic biomarkers or therapeutic targets for HCC, particularly those relevant to MAFLD-HCC. In Aim 1, we will test the hypothesis that HAF activates the NF-κB pathway by modulating the stability of TAK1 and NEMO, and thus identify the molecular mechanisms regulating the HAF- NF-κB axis. In Aim 2, we will test the hypothesis that HAF protects cells from excessive cell death during hepatitis, thus preventing progression to NASH and HCC. In Aim 3, we will test the hypothesis that HAF der...