20-hydroxyecdysone suppresses bladder cancer progression via inhibiting USP21: A mechanism associated with deubiquitination and degradation of p65

•20-hydroxyecdysone inhibits the proliferation and induces apoptosis in bladder cancer cells.•20-hydroxyecdysone suppresses the migration and invasion in bladder cancer cells.•20-hydroxyecdysone reduces tumor growth in the xenograft mouse model of bladder cancer. Bladder cancer is one of the most common malignancies of the urinary tract and a prevalent cancer worldwide, still requiring efficient therapeutic agents and approaches. 20-Hydroxyecdysone (20-HE), a steroid hormone, can be found in insects and few plants and mediate numerous biological events to control the progression of varying diseases; however, its impacts on bladder cancer remain unclear. In the study, we found that 20-HE treatments effectively inhibited the viability and proliferation of bladder cancer cells and induced apoptosis by activating Caspase-3. The migratory and invasive potential of bladder cancer cells was markedly repressed by 20-HE in a dose-dependent manner. The inhibitory effects of 20-HE on bladder cancer were confirmed in an established xenograft mouse model, as indicated by the markedly reduced tumor growth rates and limited lung and lymph node metastasis. High-throughput RNA sequencing was performed to explore dysregulated genes in bladder cancer cells after 20-HE treatment. We identified ubiquitin-specific protease 21 (USP21) as a key deubiquitinating enzyme for bladder cancer progression and a positive correlation between USP21 and nuclear factor-κB (NF-κB)/p65 in patients. Furthermore, 20-HE treatments markedly reduced USP21 expression, NF-κB/p65 mRNA, stability and phosphorylated NF-κB/p65 expression levels in bladder cancer cells, which were validated in animal tumor tissues. Mechanistic studies showed that USP21 directly interacted with and stabilized p65 by deubiquitinating its K48-linked polyubiquitination in bladder cancer cells, which could be abolished by 20-HE treatment, contributing to p65 degradation. Finally, we found that USP21 overexpression could not only facilitate the proliferation, migration, and invasion of bladder cancer cells, but also significantly eliminated the suppressive effects of 20-HE on bladder cancer. Notably, 20-HE could still perform its anti-tumor role in bladder cancer when USP21 was knocked down with decreased NF-κB/p65 expression and activation, revealing that USP21 suppression might not be the only way for 20-HE during bladder cancer treatment. Collectively, all our results clearly demonstrated that 20-HE may function as a promisi