USP28 knockdown and small molecule inhibitors promote KRT1 destabilization and sensitize hepatocellular carcinoma cells to sorafenib.

Background: Hepatocellular carcinoma (HCC) is a significant malignant tumor that is typically diagnosed late and has a poor prognosis. USP28 (Ubiquitin-specific protease 28), a deubiquitinating enzyme within the ubiquitin-specific proteases (USPs) family, plays a pivotal role in various biological processes, especially in cancer progression. However, its functions and molecular mechanisms in HCC are still unknown.

Methods: We first analyzed the expression level of USP28 in HCC tissues relative to normal tissues using TCGA database. This was further validated by qRT-PCR and Western Blot. To investigate the function of USP28 in HCC, CCK-8 assay, clone formation assay and Transwell assay were performed in control and USP28 knockdown or overexpressed HCC cells. To explore potential downstream targets of USP28, we used IP-MS analysis. The interaction between USP28 and KRT1 was confirmed by immunoprecipitation and immunofluorescence staining. Finally, we evaluated the in vivo effects of USP28 on HCC growth and metastasis using a ectopic tumor-bearing mouse model.

Results: The expression of USP28 in HCC tissues was significantly higher than that in normal tissues, and its high expression was associated with poor prognosis. Functional experiments showed that down-regulation of USP28 expression effectively inhibited the proliferation, migration and invasion of HCC cells, while overexpression of USP28 produced the opposite effect. Mechanistic investigations demonstrated that USP28 interacted with KRT1 and exerted deubiquitination on KRT1, thereby maintaining the stability of KRT1. Further studies revealed that USP28 knockdown resulted in decreased IFITM3 expression, which inhibited HCC cell proliferation. In addition, USP28 knockdown combined with sorafenib inhibited tumor growth and metastasis in tumor xenograft mice model.

Conclusions: Our study confirmed the carcinogenic effects of USP28 by stabilizing KRT1 expression and promoting IFITM3. USP28 small molecule inhibitors can inhibit the proliferation of hepatocellular carcinoma cells and enhance the sensitivity of hepatocellular carcinoma cell lines to sorafenib. This provides a theoretical basis for USP28 to be a new clinical method to alleviate sorafenib resistance.