Ubiquitination-specific protease 7 enhances stemness of hepatocellular carcinoma by stabilizing basic transcription factor 3.

This study aims to explore the molecular regulation mechanism of ubiquitination-specific protease 7 (USP7) in facilitating the stemness properties of hepatocellular carcinoma (HCC). Gain-of-function and loss-of-function assays were conducted in SK-Hep1 and HepG2 cells transfected with USP7 overexpression/knockdown plasmids and USP7 inhibitor P22077. The proliferation, migration, invasion, and self-renewal capacity of hepatocellular carcinoma cells were detected by CCK-8, colony formation, Transwell, scratch, and tumor sphere formation, respectively. MS was performed to identify the potential substrate of USP7 following P22077 treatment. Co-IP assay was used to verify the interaction between USP7 and basic transcription factor 3 (BTF3) in HCC cells. The overexpression of USP7 could promote the proliferation, migration, invasion, and colony formation capacity of SK-Hep1 and HepG2 cells. Additionally, ectopic UPS7 enhanced the epithelial-mesenchymal transition (EMT) and stem-like characteristics of the HCC cells. In contrast, USP7 depletion by knockdown of USP7 or administrating inhibitor P22077 significantly inhibited these malignant phenotypes of SK-Hep1 and HepG2 cells. Following MS analysis, BTF3 was identified as a potential substrate for USP7. USP7 could interact with BTF3 and upregulate its protein level, while USP7 depletion significantly upregulated the ubiquitination levels. Overexpression of BTF3 partially rescue the inhibitory effects of USP7 depletion on the malignant phenotypes and stemness properties of SK-Hep1 and HepG2 cells. USP7 can promote the stemness and malignant phenotype of HCC by stabilizing BTF3.

Ubiquitin-specific protease 1 facilitates hepatocellular carcinoma progression by modulating mitochondrial fission and metabolic reprogramming via cyclin-dependent kinase 5 stabilization.

Although deubiquitinases (DUBs) have been well described in liver tumorigenesis, their potential roles and mechanisms have not been fully understood. In this study, we identified ubiquitin-specific protease 1 (USP1) as an oncogene with essential roles during hepatocellular carcinoma (HCC) progression. USP1, with elevated expression levels and clinical significance, was identified as a hub DUB for HCC in multiple bioinformatics datasets. Functionally, USP1 overexpression significantly enhanced the malignant behaviors in HCC cell lines and spheroids in vitro, as well as the zebrafish model and the xenograft model in vivo. In contrast, genetic ablation or pharmacological inhibition of USP1 dramatically impaired the phenotypes of HCC cells. Specifically, ectopic USP1 enhanced aggressive properties and metabolic reprogramming of HCC cells by modulating mitochondrial dynamics. Mechanistically, USP1 induced mitochondrial fission by enhancing phosphorylation of Drp1 at Ser616 via deubiquitination and stabilization of cyclin-dependent kinase 5 (CDK5), which could be degraded by the E3 ligase NEDD4L. The USP1/CDK5 modulatory axis was activated in HCC tissues, which was correlated with poor prognosis of HCC patients. Furthermore, Prasugrel was identified as a candidate USP1 inhibitor for targeting the phenotypes of HCC by an extensive computational study combined with experimental validations. Taken together, USP1 induced malignant phenotypes and metabolic reprogramming by modulating mitochondrial dynamics in a CDK5-mediated Drp1 phosphorylation manner, thereby deteriorating HCC progression.