泛素连接酶NEDD4调控选择性自噬的机理及其在肺癌发生发展中的作用

Selective autophagy plays essential roles in regulation of metabolic and oxidative stress and multiple pathological processes. Ubiquitination has been demonstrated as a crucial biochemical modification for autophagosomal cargo recognition and recruitment in selective autophagy. However, the role of ubiquitination in regulation of autophagy receptor function remains largely unexplored. Our recent studies have found that the HECT E3 ubiquitin ligase NEDD4 interacts with the key autophagosomal protein LC3 and autophagy receptor SQSTM1 (also named as p62) and ubiquitinates SQSTM1. Knockdown of NEDD4 impaired starvation- or rapamycin-induced autophagy, significantly reduced formation of autophagosomes, and caused aggregation of LC3- and SQSTM1-positive autophagosomes in cells. Furthermore, NEDD4 is also required for lung cancer cell proliferation and migration. These data indicate an important role of NEDD4 in selective autophagy and raise a possible connection between the NEDD4-dependent ubiquitination of SQSTM1and lung tumor growth and progression. However, the mechanism and signaling pathways regulating selective autophagy by NEDD4 and the connection between selective autophagy and lung cancer are largely unknown. Thus, this proposal is to determine the molecular mechanism by which NEDD4 regulates selective autophagic processes, the lung cancer cell proliferation and migration, and lung tumor growth and metastasis through ubiquitination of SQSTM1. Our working hypothesis is that NEDD4 modifies the molecular structure of SQSTM1 by ubiquitination for facilitating interaction of SQSTM1 with its down-stream signaling molecules, activating selective autophagic process, promoting lung tumor growth and metastasis. To test the hypothesis, four specific aims will be investigated: (1) to determine the role of the NEDD4-mediated ubiquitination in regulating molecular structure and function of SQSTM1; (2) to determine the role of the NEDD4-mediated ubiquitination of SQSTM1 in regulating localization and transport of autophagosomes; (3) to determine the role of the NEDD4-mediated ubiquitination of SQSTM1 in facilitating lung cancer cell proliferation and migration; and (4) to determine the effect of NEDD4-mediated ubiquitination of SQSTM1 on xenograft tumor growth and metastasis in a nude mouse model. Through these studies, we expect to elucidate the molecular mechanism by which NEDD4 regulates selective autophagy by ubiquitination of SQSTM1 and define a new ubiquitination signaling pathway promoting lung tumorigenesis and progression.

选择性自噬在细胞能量代谢、应激反应、细胞生存等过程中起重要的调控作用。我们前期研究发现肺癌细胞中泛素连接酶NEDD4与自噬体蛋白LC3及自噬受体SQSTM1相互作用并泛素化SQSTM1。敲低NEDD4导致肺癌细胞选择性自噬体形成受阻和功能缺陷，并抑制细胞增殖和迁移。本项目基于这些新的发现，将从四个方面对NEDD4通过泛素化SQSTM1调控选择性自噬以及肺癌发生发展进行系统的研究：（1）确定该泛素化对SQSTM1分子结构功能的调节机理；（2）确定该泛素化对选择性自噬前体及自噬体形成和功能的调控机理；（3）确定该泛素化在肺癌细胞增殖和迁移中的作用；（4）确定该泛素化在裸鼠肺癌细胞移植肿瘤生长和转移中的作用。通过这些研究，揭示NEDD4泛素化信号调控选择性自噬的分子机制，确定NEDD4介导的SQSTM1泛素化在肺癌肿瘤生物学中的作用，为肺癌靶向治疗提供新的靶标途径。

本项目旨在研究自噬调节肺癌细胞功能的分子机理以及在肺癌发生发展中的作用。经过四年的努力探索，我们基本完成了研究内容和目标，并扩展了项目外的研究工作，取得了突破性的研究成果。我们的研究突破主要包括四个方面：（1）筛选和鉴定了肺癌细胞的自噬蛋白组，发现了大量新的自噬候选蛋白，为开拓自噬研究的新课题奠定了基础；（2）发现高尔基体自噬调节高尔基体结构以及肺癌细胞的增殖和迁移；（3）发现激酶NEK9通过与自噬体蛋白相互作用抑制肺癌细胞迁移；（4）确定了核受体辅助活化蛋白NCOA7与自噬体蛋白LC3的相互作用以及NCOA7在肺癌细胞中的抗氧化损伤作用。另外，我们发现了新的NEDD4调节肺癌细胞增殖迁移的信号途径以及NEDD4调节SQSTM1二聚体形成的机制。同时，我们的扩展研究工作发现了双香叶基化和YAP/TAZ调节肺癌、乳腺癌和胃癌细胞增殖和迁移的新信号途径并建立了乳腺癌和胃癌转移及不良预后的标志分子。项目负责人在本项目中以通讯作者共发表SCI学术论文10篇。本项目获得发明专利授权一项；江苏省2022年度科学技术二等奖一项；同时培养了研究生14名，其中博士研究生4名，硕士研究生10名。