USP37去泛素化修饰LSD1促进口腔鳞癌侵袭转移的机制研究

Tumor invasion and metastasis cascade is a key aspect dictating patient prognosis during oral squamous cell carcinoma progression, which also represents a significant challenge in the clinic. Our previous findings have revealed that LSD1 is a novel and pivotal mediator governing oral cancer invasion and metastasis. USP37 was initially identified as a protein modifying enzyme via in vitro deubiquitinase siRNA screen, which might regulate LSD1 protein stability and have incompletely known functions in oral cancer. Our preliminary experiments have indicated that the half-time of LSD1 protein in oral cancer cells is significantly longer than that in normal oral epithelial cells. USP37 knockdown remarkably reduced the half-time of LSD1 protein and its abundance, and significantly impaired migratory potential and invasiveness of oral cancer cells. Moreover, LSD1 protein reduction followed by USP37 knockdown was largely attenuated by MG132 addition in vitro. Here, we aim at the regulatory mechanisms underlying oral cancer invasion and metastasis by focusing on the roles of LSD1 deubiquitination via USP37. Experiments at the molecular, histological and in vivo animal levels including gene knockdown, ectopic overexpression, rescue experiment, protein immunoprecipitation, protein deubiquitination as well as deletion mapping were designed and scheduled to unravel the molecular mechanisms mediated by USP37-LSD1 regulatory axis during oral cancer invasion and metastasis. These findings might provide novel molecular biomarkers and therapeutic targets for clinical diagnosis and treatment against oral squamous cell carcinoma metastasis.

肿瘤侵袭转移是口腔鳞癌发展过程中影响患者预后的主要环节，也是当前临床诊疗的重大挑战。前期研究发现：LSD1是调控口腔鳞癌侵袭转移新的关键分子；USP37是通过去泛素化酶 siRNA library体外筛选获得、能调控LSD1蛋白稳定性但功能尚未完全知晓的蛋白修饰酶。预实验发现：LSD1蛋白在口腔癌细胞中半衰期显著长于正常口腔上皮细胞；USP37沉默后LSD1蛋白半衰期变短、表达下调，细胞侵袭转移能力明显受到抑制；USP37沉默引起的LSD1蛋白表达下调能被MG132逆转等。因此，本项目拟以口腔鳞癌侵袭转移为切入点，以USP37对LSD1蛋白的去泛素化修饰为研究对象，在体外分子水平、组织水平和活体动物水平上采用沉默、过表达、挽救实验、IP、蛋白去泛素化和缺失定位等技术进行实验研究，旨在阐明USP37-LSD1调控轴在口腔鳞癌侵袭转移过程中的分子机制，为口腔鳞癌转移的临床诊疗提供新的分子靶标。

肿瘤侵袭转移是口腔鳞癌发展过程中影响患者预后的主要环节。我们前期研究发现LSD1是调控口腔鳞癌侵袭转移新的关键分子；通过去泛素化酶siRNA library体外筛选获得USP37能调控口腔鳞癌细胞中LSD1蛋白的稳定性。本研究以口腔鳞癌转移这一临床难题为研究主线，以USP37对LSD1蛋白去泛素化修饰为研究切入点，通过系列体内外实验研究发现：1. USP37在口腔鳞癌标本和细胞系中呈异常高表达，其表达水平与肿瘤大小、颈淋巴转移、临床分期以及总体生存率相关，是判断患者预后的独立因素；2. USP37基因沉默后口腔鳞癌细胞侵袭迁移能力显著下降，而细胞增殖与凋亡变化较小；3. USP37基因沉默后口腔鳞癌细胞中LSD1蛋白表达下调而mRNA表达基本不变，LSD1蛋白降解速率显著加快，且这一效应能被MG132逆转； 4. 而LSD1下调后E-cadherin基因因启动子区H3K4me2富集增加而转录激活，促进细胞侵袭迁移；5. 蛋白免疫沉淀、泛素化检测实验证实USP37与LSD1蛋白发生结合，进而对其进行去泛素化修饰，增强其蛋白稳定性；6. 通过体外细胞药物筛选、细胞学实验和肿瘤荷瘤模型，揭示LSD1小分子抑制剂TCP与GSK-J1能够协同抑制口腔鳞癌增殖，促进凋亡。两药联合使用能够显著抑制肿瘤体内生长。上述研究成果阐明USP37-LSD1-E-cadherin分子调控轴在口腔鳞癌侵袭转移过程中的生物学作用及分子机制，有望为口腔鳞癌转移的临床诊疗提供新的分子标记物和潜在治疗靶点。在当前为数不多的口腔鳞癌致病相关去泛素化酶研究报道的背景下，本研究成果具有较高的临床价值和转化潜力。