癌蛋白c-SKI去泛素酶的筛选及其在胃癌中的功能和作用机制研究

Gastric cancer (GC) is one of the most frequent type of cancers in China. It has been reported that oncoprotein c-SKI is overexpressed in GC. However, little is known about the reason of c-SKI overexpression in GC. The disruption in the balance between ubiquitation and deubiquitation of c-SKI may be one of the reasons to cause c-SKI overexpression in GC. Either reduction in ubiquitation or increase in deubiqitation of c-SKI will lead to its overexpression in GC and the overexpressed c-SKI functions as an oncoprotein to promote GC development and progression. To identify the deubiquitinating enzyme (DUB) of c-SKI, we will establish an expression library that contains all known DUBs. The library will be.used to identify the DUB(s) that interact(s) with oncoprotein c-SKI by immunoprecipitation. The deubiquitination effect of the identified DUB(s) on c-SKI will be investigated by ubiquitination assay. The roles of the identified DUB(s) in regulating cell signaling pathway and in gastric cancer development/progression will be studied in vitro and in vivo, respectively. Finally, we will identify the small compounds that function as the inhibitor of c-SKI specific DUB(s) and explore the possibility to use those compounds as novel gastric cancer therapy reagents. We anticipate that this work will find some novel, uncharacterized DUB(s) that play(s) an important role in gastric cancer development/progression through regulating c-SKI and new therapeutic targets for treating gastric cancer.

胃癌是我国常见的恶性肿瘤之一，年死亡人数占全球的40%以上。癌蛋白c-SKI在胃癌中异常高表达，但导致其高表达的机制尚未阐明。c-SKI泛素化和去泛素化失衡可能是导致其高表达的原因之一。泛素化水平降低或去泛素化水平增加均使c-SKI表达水平升高，从而促进胃癌的发生、发展。本项目拟通过建立和筛选去泛素酶表达文库发现c-SKI特异性的去泛素酶；研究c-SKI特异性去泛素酶对c-SKI的去泛素化作用以及对与c-SKI相关的信号通路的影响；通过小鼠成瘤模型研究c-SKI特异性去泛素酶在胃癌发生、发展中的作用；筛选c-SKI特异性去泛素酶抑制剂，研究该抑制剂作为新的胃癌治疗药物的可能性。通过上述研究，本项目拟阐明癌蛋白c-SKI特异性去泛素酶在胃癌发生、发展中的功能及作用的分子机制，并为研发新的胃癌治疗药物奠定基础。项目的实施具有重要的理论价值和潜在的应用前景。

癌细胞一般具有瓦伯格效应，瓦伯格效应(Warburg effect)能够促进癌细胞代谢以适应癌细胞的快速增殖。M2型丙酮酸激酶(PKM2)是Warburg效应的重要调节因子。研究发现PKM2在许多癌细胞中高表达，且能被泛素化修饰。泛素化修饰是一个可逆的过程，机体内存在相应的去泛素化作用，可使蛋白质去泛素化，本论文的研究目的在于寻找可使PKM2去泛素化的酶，并研究其如何影响Warburg效应以及其在癌症发生、发展中的作用。胃癌是一种常见的恶性肿瘤，在我国属于高发癌症之一。胃癌的发病机制尚不明确，本论文主要研究胃癌细胞中PKM2的去泛素化酶，以及其在Warburg效应中的作用和对胃癌细胞代谢的影响，以期为明确胃癌的病因，以及寻找治疗胃癌的药物靶点提供理论依据。. 根据TCGA数据库分析发现，泛素特异性蛋白酶4(USP4)与PKM2均在胃癌中高表达，提示二者可能存在一定关系。实验结果证明USP4可以与PKM2相互作用，并能使PKM2去泛素化，从而稳定PKM2。构建USP4过表达，USP4 knockdown，USP4过表达而PKM2 knockdown等细胞系，通过进一步的研究明确了USP4对PKM2的调节作用，以及USP4的作用是由PKM2介导的。通过对细胞系进行功能研究，结果表明USP4能够促进胃癌细胞增殖以及促进胃癌细胞的葡萄糖吸收和乳酸产生，从而增强Warburg效应。