USP9X Promotes Hepatocellular Carcinoma Progression via Stabilization of HSP90AA1

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Ubiquitin-specific proteases (USPs) modulate tumor progression by regulating substrate protein stability. However, the mechanisms of most DUBs in HCC remain poorly understood. This study aimed to investigate the role of USP9X in promoting HCC cell proliferation, survival, migration, and invasion. Methods: Transcriptomic and clinical data of LIHC patients were obtained from TCGA to assess USP9X expression, prognosis, and pathway enrichment. USP9X expression in HCC and adjacent tissues was examined by immunohistochemistry and Western blotting. Functional assays (CCK-8, colony formation, wound-healing, Transwell) were performed following USP9X knockdown or overexpression in Lm3 and Huh7 cells. The USP9X–HSP90AA1 interaction was evaluated by Co-IP and mass spectrometry, and deubiquitination assays elucidated the mechanism. In vitro and in vivo experiments determined the effects of USP9X–HSP90AA1 signaling on HCC growth and metastasis. Results: Bioinformatic analyses revealed that USP9X expression was significantly elevated in LIHC tissues and correlated with poor prognosis. Immunohistochemistry and Western blotting confirmed higher USP9X protein levels in tumor tissues. Knockdown of USP9X inhibited proliferation and migration of Lm3 and Huh7 cells, whereas USP9X overexpression enhanced these phenotypes. Co-IP demonstrated a direct interaction between USP9X and HSP90AA1, and deubiquitination assays showed that USP9X decreased HSP90AA1 ubiquitination and increased its stability. Both in vitro and in vivo data indicated that USP9X promotes HCC progression through stabilization of HSP90AA1. Conclusion: USP9X is a tumor-promoting factor that accelerates HCC progression by stabilizing HSP90AA1, highlighting USP9X as a potential therapeutic target for HCC. Keywords: HCC, USP9X, HSP90AA1, deubiquitination, CO-IP, bioinformatics translation