靶向类泛素小分子NEDD8灭活Neddylation修饰通路抗食管癌的作用与机制研究

Identification of anti-tumor molecular targets is the basis of targeted drug research and development. Neddylation pathway promotes tumorigenesis by activating CRL ubiquitin ligases to induce the degradation of CRL tumor suppressor substrates, while targeting this pathway by activating enzyme inhibitor MLN4924 exerts remarkable anti-tumor effects. However, recent studies have shown that MLN4924 could cause drug resistance mutations in tumor cells. Therefore, it is urgent to explore new therapeutic targets aiming at neddylation pathway. Our recent studies found that the ubiquitin-like modifier NEDD8 is markedly overexpressed in esophageal cancer tissues which positively relates to poor prognosis of patients. Furthermore, knockout of NEDD8 could significantly inhibit the malignant phenotypes of esophageal cancer cells. These findings suggested that the downregulation of NEDD8 could inhibit the activity of CRL ubiquitin ligase by inactivating the neddylation pathway, resulting in the accumulation of its tumor suppressor substrates, thereby activating the mechanism of apoptosis and cell senescence and ultimately killing tumors. In this project, we will study effects of NEDD8 dysregulation on neddylation pathway and downstream tumor suppressors expression levels in esophageal cancer, in addition to evaluate the anti-tumor efficacy by targeting NEDD8 and elucidate the molecular mechanisms. The success of this project will provide scientific basis for NEDD8 as a novel anti-tumor target against neddylation pathway.

抗肿瘤分子靶点鉴定是靶向药物研发的基础。类泛素化Neddylation修饰通路可通过激活CRL(Cullin-Ring ligases)泛素连接酶引起CRL抑癌底物降解进而促进肿瘤发生，针对该通路活化酶的抑制剂MLN4924可显著杀伤肿瘤，但近期发现MLN4924可诱导肿瘤产生耐药突变，因此迫切需要鉴定该通路的新型抗癌靶点。我们发现类泛素小分子NEDD8在食管癌患者中高表达，并与其预后不良密切相关；且敲除NEDD8可显著抑制食管癌细胞恶性表型。据此我们提出假说：靶向下调NEDD8可通过灭活Neddylation通路抑制CRL泛素连接酶活性，导致其抑癌底物因降解受阻而积聚，进而激活细胞凋亡\衰老机制，最终杀伤肿瘤。本项目旨在阐明NEDD8失调对Neddylation通路活化及CRL底物表达影响，评价靶向NEDD8抗肿瘤效果并阐明其杀伤机制，为鉴定NEDD8为新型抗食管癌分子靶点提供科学依据。

抗肿瘤靶点鉴定是成功研发高效低毒分子靶向药物的基础。Neddylation修饰通路可通过激活CRL(Cullin-Ring ligases)泛素连接酶引起CRL抑癌底物降解进而促进肿瘤发生发展，是抗肿瘤药物筛选的重要靶标。我们前期研究发现，Neddylation通路中的类泛素小分子NEDD8在食管癌患者中高表达，并与其预后不良密切相关，并且下调NEDD8可显著抑制食管鳞癌细胞恶性表型。本项目旨在阐明NEDD8失调对Neddylation通路活化及CRL底物表达影响，评价靶向NEDD8抗肿瘤效果并阐明其杀伤机制，为鉴定NEDD8为新型抗食管癌分子靶点提供科学依据。.我们利用Crispr Cas9技术构建NEDD8下调的食管鳞癌细胞系，质谱组学分析发现下调NEDD8激活了一系列抑癌信号通路。体外和体内实验证明，下调NEDD8能显著抑制管鳞癌细胞的恶性表型和小鼠皮下瘤的生长。机制研究发现，靶向下调NEDD8的表达可以显著抑制Neddylation通路，通过灭活Neddylation底物CRL的E3泛素连接酶活性，导致其抑癌底物的积聚，进而诱导管鳞癌细胞发生DNA损伤、细胞周期阻滞，以及诱导管鳞癌细胞发生死亡受体DR5依赖的外源性细胞凋亡。.我们的研究系统阐明了靶向下调类泛素小分子NEDD8表达的抗食管癌效果并阐明其杀伤机制，为鉴定NEDD8作为针对Neddylation修饰通路的新型抗食管癌分子靶点提供科学依据，也为食管癌的靶向治疗提供参考。