Deubiquitinase USP5 promotes non-small cell lung cancer cell proliferation by stabilizing cyclin D1

Background: Cyclin D1 (CCND1) is overexpressed in non-small cell lung cancer (NSCLC) and contributes to its tumorigenesis and progression. Accumulating evidence shows that ubiquitin-specific protease 5 (USP5), an important member of the USP family, acts as a tumor promoter by deubiquitinating and stabilizing oncoproteins. However, neither the mechanism for dysregulated turnover of CCND1 protein nor the association of CCND1 with USP5 in NSCLC is well understood. Methods: The association of USP5 with CCND1 in human NSCLC cells and clinical tissues was determined by immunoprecipitation/immunoblotting, immunohistochemistry (IHC), and The Cancer Genome Atlas database analyses. The effect of USP5 knockdown or overexpression on NSCLC cell proliferation in vitro was assessed by Cell Counting Kit-8, flow cytometry-based cell cycle, and colony formation assays. The effect of the USP5 inhibitor EOAI3402143 (G9) on NSCLC proliferation in vitro was analyzed by CCK-8 assay. The effect of G9 on NSCLC xenograft tumor growth was also examined in vivo, using athymic BALB/c nude mice. Results: USP5 physically bound to CCND1 and decreased its polyubiquitination level, thereby stabilizing CCND1 protein. This USP5-CCND1 axis promoted NSCLC cell proliferation and colony formation. Further, knockdown of USP5 led to CCND1 degradation and cell cycle arrest in NSCLC cells. Importantly, this tumor-suppressive effect elicited by USP5 knockdown in NSCLC cells was validated in vitro and in vivo through chemical inhibition of USP5 activity using G9. Consistently, G9 downregulated the protein levels of CCND1 in NSCLC cells and xenograft tumor tissues. Also, the expression level of USP5 was positively associated with the protein level of CCND1 in human clinical NSCLC tissues. Conclusions: This study has provided the first evidence that CCND1 is a novel substrate of USP5. The USP5-CCND1 axis could be a potential target for the treatment of NSCLC.