E3泛素连接酶NEDL2泛素化修饰PTEN参与肺癌发生的分子机制研究

In our previous studies, we screened the human E3 ubiquitin ligase library and confirmed lung cancer related E3 ubiquitin ligase, NEDD4-like E3 ubiquitin-protein ligase 2 (NEDL2). Lung cancer cells (A549 and H1299 cells) had higher NEDL2 expression than pulmonary epithelial cells. We also observed that overexpression of NEDL2 in human lung cancer cells significantly promoted the cell proliferation and invasion. NEDL2 expression markedly increased in human lung cancer tissues. Furthermore, we demonstrated, for the first time, that phosphatase and tensin homolog (PTEN) was a substrate of NEDL2 and NEDL2-mediated ubiquitination of PTEN induced the translocation of AKT1 from cytoplasm to nucleus. These preliminary data suggest that NEDL2 plays an important role in the oncogenesis of lung cancer. In this proposed study, we will further determine the impact and underlying mechanism of NEDL2-mediated ubiquitination of PTEN on the proliferation, apoptosis, migration, and invasion of lung cancer cells. We will investigate the relationship between NEDL2 expression and the phenotype and genotype of the lung cancer patients, and define the role of NEDL2 in the development of lung cancer in animal model. The proposed studies will contribute to a greater understanding of the role of NEDL2-PTEN-PI3K-AKT signaling pathway in tumorigenesis of lung cancer and discover potential novel therapeutic targets for treating lung cancer.

为进一步阐述肺癌的发生机制，课题组前期利用蛋白筛选实验在人肺癌细胞中成功筛选并鉴定到肺癌敏感的E3泛素连接酶NEDL2；NEDL2在肺癌细胞的表达（mRNA和蛋白质水平）较肺上皮细胞明显升高，并显著促进人肺癌细胞的增殖和侵袭；与癌旁组织相比，肺癌患者肿瘤组织中NEDL2表达水平也明显升高；并首次证实PTEN是NEDL2的泛素化底物；NDEL2泛素化PTEN显著增加AKT1入核。据此我们提出科学假设：NEDL2通过泛素化修饰PTEN引起PI3K-AKT信号通路异常活化，参与肺癌的发生。本项目将进一步分析NEDL2泛素化修饰PTEN的分子机制及其对肺癌细胞生物学行为的影响；在不同类型肺癌肿瘤组织中分析NEDL2表达水平及其与患者表型和基因型的关系；并在动物模型中分析NEDL2在肺癌发生中的作用。研究结果对于进一步完善PI3K-AKT信号通路的结构，阐述肺癌发生的分子机制具有重要科学意义。

课题组在前期利用蛋白筛选实验成功筛选并鉴定到肺癌敏感的E3泛素连接酶NEDL2的基础上，证实过表达NEDL2 显著促进人肺癌A549细胞和H1299细胞的增殖、迁移和侵袭能力，而抑制内源性NEDL2显著抑制人肺癌A549细胞和H1299细胞的增殖、迁移和侵袭能力；但过表达或抑制内源性NEDL2不影响人肺癌A549细胞和H1299细胞的凋亡。NEDL2在肺癌肿瘤组织中的表达水平显著高于正常组织，并且在I期肺癌组织标本中表达变化最显著。NEDL2表达水平还与病灶组织的基因型相关，携带EGFR基因突变的组织中NEDL2的表达水平最高，提示NEDL2表达与EGFR基因突变有一定的相关性。为了分析NEDL2调控肺癌的分子机制，课题组还利用分子生物学实验首次证实PTEN是NEDL2的泛素化底物，并进一步确定NEDL2以K48链接的泛素化链形式修饰PTEN，诱导其降解，增加AKT1的入核。NEDL2泛素化修饰PTEN引起的PI3K-AKT信号通路活化效应强于该信号通路激活剂IGF-1。动物模型的结果证实NEDL2增强人肺癌细胞在裸鼠皮下成瘤的能力。研究结果进一步完善了PI3K-AKT信号通路的结构，并进一步阐述了肺癌发生的分子机制。