Abstract：
OBJECTIVE: USP14 (Ubiquitin-specific-processing protease 14) is a deubiquitinating enzyme with oncogenic effects in oral squamous cell carcinoma (OSCC). This study aims to identify new substrates of USP14 and elucidate their role in modulating cancer stem-like cells (CSCs) in OSCC.
METHODS: Bioinformatics prediction and docking were performed using UbiBrowser 2.0 and HDOCK, respectively. OSCC cell lines and patient-derived cells were used for experimental validation, employing co-immunoprecipitation, cycloheximide chase assays, and tumor sphere formation to evaluate the effects of USP14 on SOX2 stability, ubiquitination, and CSC phenotypes.
RESULTS: USP14 upregulation was associated with worse overall survival and progression-free interval in OSCC. USP14 interacted with SOX2 with its ubiquitin carboxyl-terminal hydrolase domain. USP14 knockdown impaired SOX2 stability by increasing its polyubiquitination. Ectopic overexpression of wild-type USP14, but not the hydrolase-deficient-mutant USP14C114A , enhanced SOX2 stability by reducing polyubiquitination. USP14 knockdown suppressed OSCC cell proliferation, colony formation, and tumor sphere formation in vitro and tumor growth in vivo. However, the reduction of CSC markers following USP14 knockdown was mitigated by overexpressing SOX2. These findings were verified in OSCC patient-derived CSC cells.
CONCLUSIONS: This study revealed a USP14-SOX2 axis regulating the CSC properties of OSCC.
METHODS: Bioinformatics prediction and docking were performed using UbiBrowser 2.0 and HDOCK, respectively. OSCC cell lines and patient-derived cells were used for experimental validation, employing co-immunoprecipitation, cycloheximide chase assays, and tumor sphere formation to evaluate the effects of USP14 on SOX2 stability, ubiquitination, and CSC phenotypes.
RESULTS: USP14 upregulation was associated with worse overall survival and progression-free interval in OSCC. USP14 interacted with SOX2 with its ubiquitin carboxyl-terminal hydrolase domain. USP14 knockdown impaired SOX2 stability by increasing its polyubiquitination. Ectopic overexpression of wild-type USP14, but not the hydrolase-deficient-mutant USP14C114A , enhanced SOX2 stability by reducing polyubiquitination. USP14 knockdown suppressed OSCC cell proliferation, colony formation, and tumor sphere formation in vitro and tumor growth in vivo. However, the reduction of CSC markers following USP14 knockdown was mitigated by overexpressing SOX2. These findings were verified in OSCC patient-derived CSC cells.
CONCLUSIONS: This study revealed a USP14-SOX2 axis regulating the CSC properties of OSCC.
摘要：
目的：USP14（泛素特异性加工蛋白酶14）是一种去泛素化酶，在口腔鳞状细胞癌（OSCC）中具有致癌作用。这项研究旨在鉴定USP14的新底物，并阐明它们在调节OSCC中的癌症干细胞样细胞（CSC）中的作用。
方法：使用UbiBrowser2.0和HDOCK进行生物信息学预测和对接，分别。OSCC细胞系和患者来源的细胞用于实验验证，采用免疫共沉淀，环己酰亚胺追踪试验,和肿瘤球形成，以评估USP14对SOX2稳定性的影响，泛素化，和CSC表型。
结果：在OSCC中，USP14上调与较差的总生存期和无进展间期相关。USP14通过其泛素羧基末端水解酶结构域与SOX2相互作用。USP14敲低通过增加其聚泛素化来损害SOX2的稳定性。野生型USP14的异位过表达，而不是水解酶缺陷突变型USP14C114A，通过减少聚泛素化来增强SOX2的稳定性。USP14敲低抑制OSCC细胞增殖，菌落形成,和体外肿瘤球形成和体内肿瘤生长。然而,USP14敲低后CSC标志物的减少通过过度表达SOX2得到缓解。这些发现在OSCC患者来源的CSC细胞中得到证实。
结论：本研究揭示了USP14-SOX2轴调节OSCC的CSC特性。
方法：使用UbiBrowser2.0和HDOCK进行生物信息学预测和对接，分别。OSCC细胞系和患者来源的细胞用于实验验证，采用免疫共沉淀，环己酰亚胺追踪试验,和肿瘤球形成，以评估USP14对SOX2稳定性的影响，泛素化，和CSC表型。
结果：在OSCC中，USP14上调与较差的总生存期和无进展间期相关。USP14通过其泛素羧基末端水解酶结构域与SOX2相互作用。USP14敲低通过增加其聚泛素化来损害SOX2的稳定性。野生型USP14的异位过表达，而不是水解酶缺陷突变型USP14C114A，通过减少聚泛素化来增强SOX2的稳定性。USP14敲低抑制OSCC细胞增殖，菌落形成,和体外肿瘤球形成和体内肿瘤生长。然而,USP14敲低后CSC标志物的减少通过过度表达SOX2得到缓解。这些发现在OSCC患者来源的CSC细胞中得到证实。
结论：本研究揭示了USP14-SOX2轴调节OSCC的CSC特性。
