PI3K Vps34在细胞自噬及肿瘤发展中的功能调控研究

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis, controlled by the class III PI3K complex and the lipid product of catalytic subunit VPS34, PtdIns3P. The lipid-kinase activity of VPS34 requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. A growing list of effectors might also regulate the catalytic activity of VPS34 via Beclin 1. Importantly, it has more recently been implicated that the levels of VPS34 expression is correlated with the tumorigenic activity of human breast cancer cells. However, it is still controversial in VPS34 signaling contributing to the development and progression of autophagy as well as human cancers..In this proposal, the overall hypothesis is that the active caspase 8 and Ras-like small GTPase Rasal2 plays an indispensable role in regulating the function of VPS34 and also, VPS34 itself might be a transcriptional modulator of p62 during autophagy and tumor progression. The long-term goal of this project is to obtain a greater mechanistic understanding of how nutrients such as amino acids and glucose regulate VPS34 signaling in cancer. Particular emphasis will be devoted to detailed mutual interaction analysis of VPS34 with the GAP protein Rasal2 via Gai3 under stress and to the elucidation of how cleavage of VPS34 by active caspase 8 alters its activity. Additionally, genes associated with tumor development are transcriptional regulated by VPS34 will be rigorously identified. Given the importance of VPS34 signaling in tumor development, a thorough understanding of VPS34 function will not only increase our knowledge of Class III PI3K in the regulation of both autophagy and VPS34-driven tumor progression, but will contribute directly to our overall understanding of cellular responses to changing nutritional status.

癌症的发生通常与VPS34及其产物PtdIns3P控制的自噬、 细胞内吞或胞质分裂发生障碍有关，VPS34与VPS15和Beclin 1形成复合物发挥脂质激酶活性，许多分子通过Beclin 1调控VPS34活性。虽然VPS34 表达水平与乳腺癌细胞致瘤能力相关，VPS34与自噬及癌症发生发展的关系仍有争议。本项目提出，活性caspase 8 和 Ras-like small GTPase Rasal2对VPS34在细胞感应营养变化的功能具有不可或缺的调控作用，同时VPS34可能具有转录调控的功能。本项目的长期目标是，获得对氨基酸、 葡萄糖等营养物质如何调控VPS34在自噬及癌症中的信号转导。特别注重GAP蛋白Rasal2在外界刺激下 通过Gai3 和VPS34相互作用，并揭示caspase 8调控VPS34稳定性和活性的机制；此外，还将鉴定VPS34参与肿瘤发生发展相关基因的转录调控。

自噬是细胞在应激状态下的一种应答机制，是一种进化上保守的溶酶体降解过程。通过诱导自噬能够抑制细胞凋亡的发生。相反，抑制自噬能够促进细胞凋亡和抑制肿瘤的发生。Vps34是自噬过程中必不可少的一个关键蛋白，然而我们发现Vps34除了能够诱导自噬之外，还能够通过激活p62，促进肿瘤的发生和发展。.p62是自噬过程中的一个底物蛋白，主要功能是清除泛素化蛋白和错误折叠的蛋白质，在此过程中p62本身也通过自噬途径降解。因此p62蛋白水平的升高提示自噬水平受到了抑制。在细胞内，p62蛋白不同的位点都能够磷酸化，而p62通常处于这些磷酸化状态的动态平衡中。最近报道显示，p62的UBA区域的S403被磷酸化能够增加p62与错误折叠蛋白的亲和力并促进该蛋白的降解。我们发现在MCF-7细胞中，p62蛋白转录水平是受Vps34调控的。vps34招募PKCδ,诱导p62分子表达，然而该p62分子主要存在S349 磷酸化状态，从而促进癌症的发生和发展。尽管Vps34在自噬中起关键作用，我们的研究表明Vps34也能够通过激活MEK-ERK通路，调控癌细胞生长。进一步的研究发现，p62也是激活MEK-ERK的关键因子，因此p62是Vps34诱导的癌症发生和发展中的关键作用。基于以上结论，我们提出相比于自噬抑制肿瘤的生长，自噬关键蛋白Vps34能够通过激活p62促进肿瘤的发生和发展。表明 Vps34不仅是一个自噬的重要调节分子，也是一个肿瘤发生和发展的潜在新靶标。本课题为更深入的探究Vp34的功能以及在肿瘤中的具体的分子作用机制提供一定的理论基础，也为改善胰乳腺癌治疗提供新的方法和策略，同时助于进一步完善以Vps34 为靶标的抗肿瘤药物研发，具有重要的理论意义和临床意义。