USP5 promotes tumor progression by stabilizing SLUG in bladder cancer

Abstract

Bladder cancer ranks as the second most prevalent urology malignancy globally. Invasive metastasis is a significant contributor to mortality among patients with bladder cancer, yet the underlying mechanisms remain elusive. Deubiquitinases are pivotal in carcinogenesis, with USP5 implicated in the malignant progression of hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer. The present study assessed the role and mechanism of ubiquitin-specific proteinase 5 (USP5) in the malignant progression of bladder cancer. The association between USP5 expression and bladder cancer prognosis and stage was analyzed using The Cancer Genome Atlas database. Moreover, to elucidate the role of USP5 in bladder cancer, USP5 overexpression and knockdown cell lines were established using T24 cells. Cell viability, proliferation and migration were assessed using Cell Counting Kit-8, Transwell and scratch assays, respectively. Cyclohexanamide was used to evaluate the effect of USP5 expression on Snail family zinc finger 2 (SLUG) stability. Immunoprecipitation and immunofluorescence co-localization were utilized to probe the interaction between USP5 and SLUG. Changes in mRNA and protein levels were assessed using reverse transcription-quantitative PCR and western blotting, respectively. The results revealed that patients with bladder cancer with high USP5 expression had significantly shorter survival (P<0.05) and a higher clinicopathologic stage (P<0.05) than those with low USP5 expression. T24 cells overexpressing USP5 demonstrated significantly increased proliferation (P<0.05), invasion (P<0.05) and expression of epithelial-mesenchymal transition markers (P<0.05); whereas T24 cells with knocked-down USP5 expression revealed significantly reduced proliferation (P<0.05), invasion (P<0.05) and epithelial-mesenchymal transition markers (P<0.05). Immunoprecipitation experiments demonstrated the binding of USP5 to SLUG in bladder cancer cells, with further analysis revealing that USP5 upregulated protein levels of SLUG by inhibiting its ubiquitination. Furthermore, the treatment of bladder cancer cells with Degrasyn, a USP5 inhibitor, was associated with a significant inhibition of the proliferation (P<0.05) and invasion (P<0.05) of T24 cells. In conclusion, the findings of the present study underscore the role of USP5 in promoting the malignant progression of bladder cancer through the stabilization of SLUG. Targeting USP5 holds promise as a strategy for inhibiting bladder cancer progression.

Keywords: Snail family zinc finger 2; bladder cancer; epithelial-mesenchymal transition; ubiquitin-specific proteinase 5.

Figure 1.

USP5 expression is associated with tumor progression in patients with bladder cancer. (A) Relationship between the USP5 expression level and overall survival using data from TCGA-BLCA dataset. Analysis of USP5 expression in different (B) T and (C) N stages using data from TCGA-BLCA dataset. (D) Correlation analysis of USP5 with epithelial-mesenchymal transition markers (CDH2, ACAT2 and VIM) using data from TCGA-BLCA dataset. (E) Representative immunohistochemistry staining images of USP5 in normal bladder tissue, NMIBC and MIBC. *P<0.05. USP5, ubiquitin-specific proteinase 5; TCGA-BLCA, The Cancer Genome Atlas-Bladder cancer; T, tumor; N, node; CDH2, cadherin 2; ACAT2, acetyl-CoA acetyltransferase 2; VIM, vimentin; NMIBC, non-basal invasive bladder cancer; MICB, muscle invasive bladder cancer; TPM, transcripts per million; HR, hazard ratio; CI, confidence interval.

Figure 2.

USP5 promotes the malignant phenotype of human bladder cancer T24 cells. (A) Protein expression levels of USP5 in knockdown and overexpression human bladder cancer T24 cell lines. (B) Cell scratch test results of human bladder cancer T24 cells after knockdown and overexpression of USP5. Images were captured with a light microscope at 0 and 24 h. Results of the (C) Transwell and (D) cell proliferation assays after the knockdown and overexpression of USP5. (E) Western blot results demonstrate the expression of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin and vimentin) in human bladder cancer T24 cells after the knockdown and overexpression of USP5. *P<0.05. USP5, ubiquitin-specific proteinase 5; sh, short hairpin; NC, negative control; OD, optical density.

Figure 3.

USP5 interacts with SLUG in human bladder cancer T24 cells. (A) Protein interaction network map of proteins bound to USP5 using data from the GeneMANIA database. (B) Colocalization of USP5 and SLUG in T24 cells was visualized using a confocal microscope, and ImageJ software was used for colocalization analysis of USP5 and SLUG expression. Pearson correlation analysis revealed that r > 0.5, and Manders' colocalization coefficient demonstrated that M1 > 0.5 and M2 > 0.5. (C) Computational docking model of USP5 and SLUG. (D) Endogenous and exogenous IP demonstrated the interaction of USP5 and SLUG in T24 cells. USP5, ubiquitin-specific proteinase 5; SLUG, Snail family zinc finger 2; IP, immunoprecipitation.

Figure 4.

USP5 stabilizes SLUG by reducing its ubiquitination. (A) Protein expression and (B) mRNA levels of SLUG in the USP5 knockdown and overexpression T24 cell lines. (C) Remaining SLUG protein levels in the USP5 knockdown and overexpression T24 cell lines after treatment with CHX at several time points. (D) Western blotting revealed that USP5 regulated SLUG ubiquitination in T24 cells. SLUG proteins were isolated from the USP5 knockdown or overexpression T24 cells by Co-IP, and the ubiquitination of vimentin was then detected using western blotting. *P<0.05. USP5, ubiquitin-specific proteinase 5; SLUG, Snail family zinc finger 2; IP, immunoprecipitation; CHX, cyclohexanamide; sh, short hairpin; NC, negative control; UB, ubiquitination.

Figure 5.

USP5 promotes the progression of bladder cancer by interacting with SLUG. (A) Protein expression and (B) mRNA levels of SLUG in the USP5 overexpression T24 cell lines with and without SLUG knockdown. Results of the (C) Transwell and (D) cell proliferation assays in the USP5 overexpression T24 cell line with or without SLUG knockdown. (E) Protein expression levels of epithelial-mesenchymal transition marker (E-cadherin, N-cadherin and vimentin) in the USP5 overexpression T24 cell lines with or without SLUG knockdown. *P<0.05; ^#P>0.05. USP5, ubiquitin-specific proteinase 5; SLUG, Snail family zinc finger 2; sh, short hairpin; OD, optical density.

Figure 6.

Pharmacological inhibition of USP5 suppresses bladder cancer progression. (A) Cell viability of T24 cells at different Degrasyn concentrations. (B) Protein expression and (C) mRNA levels of SLUG in the USP5 overexpression T24 cell line with or without Degrasyn treatment (USP5 inhibitor, 2 µg/ml). Results of the (D) Transwell and (E) cell proliferation assays in the USP5 overexpression T24 cell line with or without Degrasyn treatment (2 µg/ml). (F) Protein expression levels of epithelial-mesenchymal transition markers (E-cadherin, N-cadherin and vimentin) in the USP5 overexpression T24 cell line with or without Degrasyn treatment (2 µg/ml). *P<0.05; ^#P>0.05. USP5, ubiquitin-specific proteinase 5; SLUG, Snail family zinc finger 2; IC₅₀, half maximal inhibitory concentration; OD, optical density.