CBX4通过SUMO化SKIL促进食管鳞癌细胞上皮间质转化的机制研究

Esophageal squamous cell carcinoma(ESCC) is one of the most common malignancies with high mortality, and there is no effective targeted treatment until now. Our preliminary study found that CBX4(Chromobox Homolog 4), a SUMO E3 ligase, had significant copy number amplifications in ESCC. Further investigation indicated that CBX4 could promote the proliferation, invasion and migration of ESCC, and may play a role of oncogene. Besides, the results of CoIP and LC-MS/MS illustrated that CBX4 was closely associated with SKIL, the inhibitor of TGF-β/SMADs pathway. Therefore, we speculate that CBX4 may degrade SKIL by SUMOlyation to activate TGF-β pathway, induce epithelial-mesenchymal transition, and enhance the invasion and migration of ESCC. In view of the hypothesis above, this proposal aims to reveal the mechanism of CBX4 in invasion and migration of ESCC by dual-luciferase reporter assay, GST Pull-down and in vitro experiments. In addition, we explore the correlation between the expression of CBX4 and clinical information of ESCC patients by FISH and IHC experiments. These results may reveal the mechanism of CBX4 in the development of ESCC and provide a theoretical foundation for designing effective drug targets.

食管鳞癌是我国高发病率和高致死率的恶性肿瘤，目前尚缺乏有效的靶向治疗手段。课题组前期组学测序分析发现CBX4（Chromobox Homolog 4），一种SUMO E3连接酶，在食管鳞癌中存在显著的拷贝数扩增。前期细胞学实验表明CBX4可明显促进食管鳞癌细胞增殖、侵袭和迁移，可能发挥癌基因样作用。免疫共沉淀和蛋白质谱检测提示CBX4与TGF-β/SMADs通路中的抑制因子SKIL存在相互作用，推测CBX4可能SUMO化SKIL促进其降解、激活TGF-β信号通路进而诱导细胞上皮间质转化，侵袭迁移能力增强。本项目针对该假设，拟通过蛋白互作、双荧光素酶报告系统、体外动物实验等阐述CBX4在食管鳞癌侵袭转移中的作用机制；通过FISH和IHC检测食管鳞癌大样本组织中CBX4的表达量，明确其与患者临床信息的相关性，为揭示CBX4在食管鳞癌发生发展的机制、设计有效药物靶点提供理论依据。

研究背景：食管鳞癌是我国高发病率和高致死率的恶性肿瘤，CBX4是新发现的食管鳞癌拷贝数扩增基因，但其在食管鳞癌中的作用机制和临床意义尚未知。.研究内容：统计临床样本中CBX4表达，分析其与临床信息（食管鳞癌分期分型、预后等）的相关性；在食管鳞癌细胞系中分别敲低及过表达CBX4，研究其对细胞增殖、侵袭迁移的影响；通过蛋白质谱、免疫共沉淀、GST Pull-down等研究CBX4对食管鳞癌发生发展的可能的作用机制。.结果：1.CBX4在食管鳞癌中发挥癌基因样作用，提示不良预后；2.细胞水平证明敲低CBX4后，食管鳞癌细胞增殖受限，侵袭迁移能力被抑制，动物水平提示过表达CBX4促进体内成瘤，肿瘤细胞更易发生转移；3.Western Blot实验表明CBX4可引起肿瘤细胞的上皮间质转化相关蛋白水平变化；4.蛋白质谱、免疫共沉淀和GST Pull-down试验提示CBX4与NAT10蛋白间存在互作关系；5.干扰挽救实验揭示CBX4通过调控NAT10的表达而发生上皮间质转化，进而推动食管鳞癌细胞发生转移。.意义：本研究部分揭示了CBX4在食管鳞癌发生发展中的作用及机制，有望为食管鳞癌的精准医疗提供可能的分子标志物，并为药物研发提供可能的靶点。