A Role for the Novel HAF-NFkappaB Axis in Driving Obesity-Associated Liver Cancer

肝细胞癌（Hepatocellular carcinoma，HCC）是原发性肝癌的主要病因，是第四大恶性肿瘤 死亡，以及美国增长最快的恶性肿瘤。晚期HCC在很大程度上仍然无法治愈， 反应率低（<20%）和治疗抗性。目前的肥胖流行与 代谢（功能障碍）相关性脂肪肝（MAFLD）及其炎性 非酒精性脂肪性肝炎（NASH），可导致HCC。MAFLD-HCC发病率正在增加 戏剧性地，强调了对新的诊断和治疗策略的未满足的医疗需求。这里我们 描述了缺氧相关因子HAF在MAFLD-HCC中的新的肿瘤抑制作用。全局 小鼠中HAF的单倍不足和肝细胞特异性缺失导致具有NASH特征的HCC 包括重度脂肪变性伴肝细胞变性（肝细胞气球样变）、纤维化和肝细胞增殖增加。 炎性细胞浸润。在两种小鼠模型中或通过siRNA转染HCC细胞中的HAF损失与 p65/p50 NF-κB转录亚基的活化降低， 上游调节子TAK 1和NEMO。内源性HAF与NEMO和TAK 1形成复合物，表明 HAF通过直接调节这些蛋白质的稳定性来调节NF-κB通路，可能是通过 HAF的E3泛素连接酶活性。HAF敲低与自发凋亡增加有关， 而HAF过表达保护细胞免受TNF诱导的细胞死亡，这表明HAF可能在细胞凋亡中起作用。 通过保护细胞免受与肝脏炎症（肝炎）相关的死亡， 导致HCC。事实上，HAF水平受到肝炎期间普遍存在的条件如缺氧和缺氧的抑制。 TNF或TGF-β升高，而HAF则因DNA损伤而增加，这表明HAF可能有助于 NF-κB活化对癌前肝细胞基因组不稳定性的反应值得注意的是，HAF高度 在大多数人肝炎病例中表达，但在94%的人HCC中检测不到（65例）。 因此，我们假设HAF通过促进NF-κB的活化而在HCC中发挥新的肿瘤抑制作用 在肝炎期间促进肝细胞的存活。缺氧或炎症抑制HAF 肝炎期间的细胞因子导致细胞更新增加，从而驱动向NASH和HCC的进展。我们 总体目标是鉴定HCC的新的预测/预后生物标志物或治疗靶点，特别是那些 与MAFLD-HCC相关。在目标1中，我们将检验HAF通过以下途径激活NF-κB通路的假设： 调节TAK 1和NEMO的稳定性，从而确定调节HAF的分子机制。 NF-κB轴。在目标2中，我们将检验HAF在肝炎期间保护细胞免于过度细胞死亡的假设， 从而防止进展为NASH和HCC。在目标3中，我们将检验HAF放松管制是 通过研究> 500例患者样本中的HAF表达， HAF及其下游靶点与HCC发生和进展或治疗反应的相关性。

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 deregulation is associated with progression to HCC by investigating HAF expression in > 500 patient samples, to determine the association of HAF and its downstream targets to HCC initiation and progression or to treatment response.