目的:泛素特异性肽酶10(USP10),一种典型的去泛素酶,已被发现在人类癌症中起着双重作用。以前,我们报道USP10的表达与胃壁浸润深度呈负相关,淋巴结转移,胃癌(GC)患者的预后。然而,目前尚不清楚USP10是否可以通过其去泛素化功能调节GC细胞的转移。
方法:在本研究中,蛋白质组,泛素组,并进行转录组分析以全面鉴定GC细胞中USP10的新型去泛素化靶标。随后,一系列验证实验,包括体外细胞培养研究,体内转移肿瘤模型,和临床样本分析,进行以阐明USP10及其去泛素化靶标在GC转移中的调节机制。
结果:在GC细胞中过表达USP10后,146种蛋白质,489个泛素位点,61个mRNA表现出差异表达。通过整合多组学的结果,我们最终筛选了USP10的9种潜在底物,包括TNFRSF10B,SLC2A3、CD44、CSTF2、RPS27、TPD52、GPS1、RNF185和MED16。其中,通过Co-IP和蛋白质稳定化测定进一步证实TNFRSF10B是USP10的直接去泛素化靶标。在体外和体内模型中,USP10或TNFRSF10B的失调影响了GC细胞的迁移和侵袭。分子机制研究表明,USP10通过增加TNFRSF10B蛋白的稳定性抑制上皮间质转化(EMT)过程,从而调控GC细胞的迁徙和侵袭。最后,回顾性临床样本研究表明,TNFRSF10B表达的下调与7个GC队列中的4个患者的低生存率相关,TNFRSF10B蛋白的表达与远处转移的发生率呈显著负相关,漫反射类型,和粘性差的癌。
结论:我们的研究建立了筛选USP10去泛素化靶标的高通量策略,并进一步证实抑制TNFRSF10B的泛素化可能是GC转移的有希望的治疗策略。
OBJECTIVE: Ubiquitin-specific peptidase 10 (USP10), a typical de-ubiquitinase, has been found to play a double-edged role in human cancers. Previously, we reported that the expression of USP10 was negatively correlated with the depth of gastric wall invasion, lymph node metastasis, and prognosis in gastric cancer (GC) patients. However, it remains unclear whether USP10 can regulate the metastasis of GC cells through its de-ubiquitination function.
METHODS: In this study, proteome, ubiquitinome, and transcriptome analyses were conducted to comprehensively identify novel de-ubiquitination targets for USP10 in GC cells. Subsequently, a series of validation experiments, including in vitro cell culture studies, in vivo metastatic tumor models, and clinical sample analyses, were performed to elucidate the regulatory mechanism of USP10 and its de-ubiquitination targets in GC metastasis.
RESULTS: After overexpression of USP10 in GC cells, 146 proteins, 489 ubiquitin sites, and 61 mRNAs exhibited differential expression. By integrating the results of multi-omics, we ultimately screened 9 potential substrates of USP10, including TNFRSF10B, SLC2A3, CD44, CSTF2, RPS27, TPD52, GPS1, RNF185, and MED16. Among them, TNFRSF10B was further verified as a direct de-ubiquitination target for USP10 by Co-IP and protein stabilization assays. The dysregulation of USP10 or TNFRSF10B affected the migration and invasion of GC cells in vitro and in vivo models. Molecular mechanism studies showed that USP10 inhibited the epithelial-mesenchymal transition (EMT) process by increasing the stability of TNFRSF10B protein, thereby regulating the migration and invasion of GC cells. Finally, the retrospective clinical sample studies demonstrated that the downregulation of TNFRSF10B expression was associated with poor survival among 4 of 7 GC cohorts, and the expression of TNFRSF10B protein was significantly negatively correlated with the incidence of distant metastasis, diffuse type, and poorly cohesive carcinoma.
CONCLUSIONS: Our study established a high-throughput strategy for screening de-ubiquitination targets for USP10 and further confirmed that inhibiting the ubiquitination of TNFRSF10B might be a promising therapeutic strategy for GC metastasis.