SUMO化修饰调控Snail蛋白质稳定性在NSCLC细胞EMT和转移过程中的的机制与功能研究

It has been extensively accepted that metastasis of tumor cells contributed to poor prognosis in advanced non-small cell lung cancer patients. Epithelial-mesenchymal transition (EMT) has been regarded as a key step in tumor metastasis. Our previous study has uncovered that the inhibition of FBW7, a ubiquitin E3 ligase, could stabilize Snail, a well-known iTF of EMT, and in turn to promote NSCLC invasion and migration. However, its underlying mechanism is still unclear. Our group has recently found that SUMOylation of Snail functions in mediating its recognition and combination with FBW7. This project aims to further illustrate the pivotal role of SUMO modification in mediating recognition and combination between Snail and FBW7 via bioinformatics analysis, molecular biology and animal models, and further screen the key SUMO-E3 ligase in Snail SUMOylation, uncovering its underlying functions in lung cancer metastasis. Additionally, we intend to ensure the interacting motif between FBW7 and SUMOylated Snail, explore the potential mechanism in FBW7-mediated Snail degradation, and further validate hypothesis above in clinical samples. This study will explore the effects of SUMOylation in FBW7-related substrates recognition, uncover its clinical implications, and further provide a brand new insight and target to overcome advanced NSCLC metastasis.

肿瘤细胞的全身转移是造成晚期NSCLC患者预后不佳的重要原因之一。目前认为上皮细胞间质转化（EMT）在肿瘤转移中发挥重要作用。我们前期研究发现泛素连接酶FBW7表达降低可增加EMT关键分子Snail的蛋白稳定性并促进NSCLC转移，但其分子机制尚未完全阐明。我们近期发现Snail的SUMO化修饰在介导FBW7识别与结合Snail的过程中发挥重要作用。本课题拟进一步利用生物信息分析、分子生物学及动物模型构建等方法，筛选SUMO化的关键修饰酶，明确SUMO修饰后Snail与FBW7的结合位点，探索FBW7识别和降解Snail的分子机制，阐明Snail的SUMO修饰在肺癌转移过程中的功能和机制，并在临床组织及血液样本中进行验证。本研究将解析SUMO修饰在调控FBW7底物识别中的关键作用，并揭示其临床意义，为临床治疗晚期NSCLC转移提供新的思路和靶点。

本课题主要从SUMO化修饰角度揭示SUMO化修饰的关键底物在调控肺癌发展中的潜在作用，本课题主要从以下内容展开：1.我们收集了3例肺腺癌患者的肿瘤组织及其癌旁组织并进行4DSUMO化蛋白质修饰组学分析，发现肿瘤发生与AhR等关键代谢靶点密切相关。进一步分析上述修饰位点的氨基酸序列明确关键的修饰结构域并在AhR蛋白质氨基酸序列中验证上述结构域的种属保守性。同时，我们在3个独立数据库中均发现上述AhR修饰位点的存在，即K510位点。另外，我们在临床组织中也鉴定到同样的结果，即肿瘤组织中AhR与UBC9均呈高表达状态切两者间存在一定的相关性，基于以上内容我们推测AhR可能作为UBC9介导的SUMO化修饰第五存在并与后续泛素化修饰密切关联。随后我们在肺癌细胞系中对上述分子进行、筛选并选择H1975与H460细胞系进行后续研究。给予SUMO E1抑制剂TAK-981或蛋白酶体抑制剂MG132等均可影响AhR SUMO化修饰水平。同时，AhR的SUMO化修饰主要是在其第510号位点进行。2.本课题组前期工作聚焦在FBW7介导的蛋白质泛素化修饰，我们发现FBW7与m6A甲基化修饰密切相关。因此，通过前期筛选找到FBW7与甲基转移酶METTL3的具有潜在的相关性，并在细胞与肿瘤组织中得以验证。明确METTL3作为FBW7潜在修饰底物，通过RIP等实验验证上述猜想）并在动物实验与人体组织样本中得以证实。3. 从泛素化修饰角度探索其在预测肺癌疗效、COVID-19感染后免疫系统变化及肺癌抗肿瘤免疫治疗后相关性肝转移的临床特点等。本课题旨在探索临床潜在的SUMO化修饰干预靶点，为提高肺癌患者远期生存提供理论基础，同时，从泛素化修饰角度揭示其在肿瘤发展、抗肿瘤免疫治疗及感染中的作用，为后续研究奠定基础。