Neddylation通路E2耦联酶Ube2F在促肺非小细胞癌发生中的作用和机制研究

Protein neddylation is a process catalyzed by an E1 NEDD8 Activating Enzyme (NAE), an E2 NEDD8-conjugating enzyme and an E3 NEDD8-ligase to tag NEDD8 onto a substrate protein, not for degradation, but for modulation of protein activity or function. We recently found that components of neddylation pathway, including NEDD8, NAE, E2 (Ube2M), E3 (SAG), are overactivated in lung cancer (JCI & JNCI, 2014). How abnormal protein neddylation regulates tumorigenesis, however, remains elusive. Our preliminary data showed that 1) UBE2F, a newly identified neddylation E2, is overexpressed in mouse lung tumors derived from Kras activation as well as in non-small cell lung carcinoma (NSCLC) tissues, which is associated with poor patient survival; and 2) Ube2F overexpression promotes, whereas Ube2F silencing inhibits, growth of lung cancer cells, but not immortalized nontransformed cells. We, therefore, hypothesize that Ube2F-related neddylation pathway plays a central role in lung tumorigenesis, thus serving as a promising drug target for lung cancer therapy. The goals of this application are to use cell biology, genetic mouse KO models, and proteomics approaches to fully characterize mechanistically the promoting role of Ube2F in lung tumorigenesis. Three specific aims are proposed to understand 1) Whether and how Ube2F promotes proliferation of lung cancer cells; 2) whether and how UBE2F contributes to lung tumorigenesis triggered by combination of KrasG12D activation and p53 deletion, and 3) what is the underlying mechanism by identification and characterization of novel Ube2F substrates. Our proposed study is of high significance with translational value by further validating neddylation pathway as an attractive drug target and providing fundermental basis for future development of small molecule inhibitors of Ube2F as a novel class of anti-lung cancer therapy.

探讨类泛素化异常修饰如何调控肿瘤发生及其机制是当前该领域的热点。我们发现该通路中NEDD8、NAE、E2（Ube2M）及E3（SAG）在肺癌组织中过度激活，但其诱导肺癌发生的机制尚不明确。我们近期发现，新鉴定的E2 Ube2F在人肺癌及KrasG12D诱导的小鼠肺肿瘤中过表达；敲除 Ube2F可抑制肺癌细胞增殖，而对正常细胞无影响。我们提出Ube2F相关通路在肺癌发生中发挥着重要作用并有望成为新的肺癌特异性治疗靶点。我们拟通过细胞水平及小鼠体内成瘤及转移模型明确Ube2F与肺癌发生发展的关系；用条件性敲除小鼠及相关原代细胞探讨Ube2F在Kras激活和p53失活导致肺癌形成中的作用；用蛋白组学寻找Ube2F促进肺癌发生的新的下游底物。本课题将进一步论证类泛素通路作为新的肺癌治疗靶点的可行性，为研发特异性靶向Ube2F通路的小分子抑制剂及其作为抗肺癌新药提供充分的理论依据。

蛋白拟素化修饰通路中的多个组分在肺癌临床样本都被发现存在异常活化。但是，拟素化修饰通路异常活化在肺癌发生发展中的作用还不清楚。Ube2F 是蛋白拟素化修饰的两个E2 耦联酶之一，本项目以Ube2F在肺癌发生发展中的作用为课题，在以下几方面取得了重要进展：1）阐明了Ube2F在非小细胞肺癌细胞体内和体外生长的作用：Ube2F过表达能促进肺癌细胞在体内外的生长和增殖； Ube2F敲降通过诱导细胞凋亡抑制肺癌细胞的生长；2）找到Ube2F作用的新底物NOXA，并阐明了Ube2F通过调控NOXA的降解来调控非小细胞肺癌细胞在体内外生长；3）揭示了Ube2F调控NOXA降解的分子机制，即Ube2F通过参与催化CUL5的拟素化修饰，激活CRL5泛素连接酶催化NOXA发生K11链多聚泛素化，从而促进NOXA的降解； 4）研究发现Ube2F在肺癌临床样本中过表达，且高表达的患者预后更差；在Ube2F高表达的肺癌患者中，高CUL5或者低NOXA表达的患者预后更差；5）揭示了Ube2F蛋白的泛素化和降解调控机制，即在无刺激的生理情况下，CRL3 Keap1负责Ube2F的泛素化和降解，而在刺激情况下，由Ube2M-Parkin1-DJ-1复合物促进Ube2F的泛素化和降解；6）研究发现了Ube2M或Parkin通过靶向Ube2F降解来抑制肺癌细胞生长；7）研究发现在Kras活化诱导的肺癌小鼠模型中，Ube2F敲除可促进肺部巨噬细胞浸润和炎症发生，导致小鼠窒息死亡。总之，本项目从体外细胞水平、基因敲除鼠肺癌模型和肺癌临床样本三个层次，研究了Ube2F在肺癌发生中的作用及其机制。我们利用人体外肺癌细胞和临床肺癌患者样本的研究提示Ube2F是一个理想的肺癌治疗靶点。非常有趣的发现是：在Kras活化诱导的肺癌小鼠中Ube2F敲除明显促进肺部炎症的发生，其作用机制还有待于进一步阐明。