Chang Chunxiao, Wang Meng, Li Jia, Qi Sihao, Yu Xiaojuan, Xu Jun, Shi Shengbin
Department of Gastrointestinal Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University, Shandong Academy of Medical Sciences, Jinan City, 250117, Shandong Province, China.
Department of Oncology, Shandong Jining First People's Hospital, Jining City, 272111, Shandong Province, China.
J Exp Clin Cancer Res. 2025 Aug 9;44(1):232. doi: 10.1186/s13046-025-03488-3.
BACKGROUND: Chronic liver injury is a key factor in diseases like hepatocellular carcinoma (HCC), steatohepatitis (NASH), and viral hepatitis type B and C (HBV, HCV). Understanding its molecular mechanisms is crucial for effective treatment. The NOTCH1 signaling pathway, though not fully understood, is implicated in liver injury and may be a potential therapeutic target. METHODS: Clinical HCC, HBV, HCV and NASH samples and additional in vitro and in vivo performances were subjected to confirm the role of NOTCH1 and its downstream targets via a series of biochemical assays, molecular analysis approaches and targeted signaling pathway assay, etc. RESULTS: The present study first verified the abnormal elevation of NOTCH1 in hepatocytes from patients with steatohepatitis, HCC, HBV, HCV, and mouse models. Crucially, we discovered that hepatocyte-specific NOTCH1 knockout reduces hepatocellular damage in chronic liver inflammation and HCC mouse models, whereas adeno-associated virus serotype 8 (AAV8)-mediated NOTCH1 overexpression in hepatocytes exacerbates liver injury-related phenotype on-setting. Mechanistically, we showed that NOTCH1 has a new role in controlling ferroptosis and oxidative damage in hepatocytes. It interacts with Kelch-like ECH-associated protein 1 (KEAP1) and is directly recruited through its intracellular domain (NICD1). Additionally, the KEAP1 recruited by NOTCH1 impeded the binding stability of KEAP1-NFE2 like BZIP transcription factor 2 (Nrf2), promote the separation of KEAP1 and Nrf2, thereby reducing the stability of Nrf2 and hindering the ubiquitination-related proteasome degradation of Nrf2. Crucially, we also discovered that NOTCH1's ANK domain is essential for NICD1-KEAP1 contacts and signaling activation. The inability of NOTCH1 with ANK domain mutants (ΔANK) to connect with KEAP1 and increase its expression emphasizes the importance of the ANK domain in KEAP1-NRF2 signaling. By reversing the downregulation of KEAP1 and the overexpression of NRF2, ANK function is linked to ferroptosis and ROS buildup. ANK domain targeting may slow the course of HCC and liver damage. CONCLUSIONS: Targeting the NOTCH1-KEAP1-NRF2 axis as a possible chronic hepatic injury therapy is supported by these findings, which identify NOTCH1-KEAP1 as an NRF2 suppressor that accelerates the progression of liver injury.
背景：慢性肝损伤是肝细胞癌（HCC）、非酒精性脂肪性肝炎（NASH）以及乙型和丙型病毒性肝炎（HBV、HCV）等疾病的关键因素。了解其分子机制对于有效治疗至关重要。NOTCH1信号通路虽尚未完全明确，但与肝损伤有关，可能是一个潜在的治疗靶点。 方法：对临床HCC、HBV、HCV和NASH样本以及其他体外和体内实验进行一系列生化检测、分子分析方法和靶向信号通路检测等，以证实NOTCH1及其下游靶点的作用。 结果：本研究首次证实了非酒精性脂肪性肝炎、HCC、HBV、HCV患者的肝细胞以及小鼠模型中NOTCH1异常升高。至关重要的是，我们发现肝细胞特异性NOTCH1基因敲除可减轻慢性肝脏炎症和HCC小鼠模型中的肝细胞损伤，而腺相关病毒8型（AAV8）介导的肝细胞中NOTCH1过表达会加剧肝损伤相关表型的出现。从机制上讲，我们表明NOTCH1在控制肝细胞铁死亡和氧化损伤方面具有新作用。它与 Kelch样ECH相关蛋白1（KEAP1）相互作用，并通过其细胞内结构域（NICD1）直接招募KEAP1。此外，NOTCH1招募的KEAP1阻碍了KEAP1与NFE2样BZIP转录因子2（Nrf2）的结合稳定性，促进了KEAP1与Nrf2的分离，从而降低了Nrf2的稳定性，并阻碍了Nrf2的泛素化相关蛋白酶体降解。至关重要的是，我们还发现NOTCH1的ANK结构域对于NICD1-KEAP之间的接触和信号激活至关重要。NOTCH1的ANK结构域突变体（ΔANK）无法与KEAP1连接并增加其表达，这强调了ANK结构域在KEAP1-NRF2信号传导中的重要性。通过逆转KEAP1的下调和NRF2的过表达，ANK功能与铁死亡和ROS积累相关。靶向ANK结构域可能会减缓HCC和肝损伤的进程。 结论：这些发现支持将NOTCH1-KEAP1-NRF2轴作为一种可能的慢性肝损伤治疗靶点，这些发现确定NOTCH1-KEAP1是一种加速肝损伤进展的Nrf2抑制剂。
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