Target deubiquitinase OTUB1 as a therapeatic strategy for BLCA via β-catenin/necroptosis signal pathway

Abstract

Ubiquitination, a prevalent and highly dynamic reversible post-translational modification, is tightly regulated by the deubiquitinating enzymes (DUBs) superfamily. Among them, OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 1 (OTUB1) stands out as a critical member of the OTU deubiquitinating family, playing a pivotal role as a tumor regulator across various cancers. However, its specific involvement in BLCA (BLCA) and its clinical significance have remained ambiguous. This study aimed to elucidate the biofunctions of OTUB1 in BLCA and its implications for clinical prognosis. Our investigation revealed heightened OTUB1 expression in BLCA, correlating with unfavorable clinical outcomes. Through in vivo and in vitro experiments, we demonstrated that increased OTUB1 levels promote BLCA tumorigenesis and progression, along with conferring resistance to cisplatin treatment. Notably, we established a comprehensive network involving OTUB1, β-catenin, necroptosis, and BLCA, delineating their regulatory interplay. Mechanistically, we uncovered that OTUB1 exerts its influence by deubiquitinating and stabilizing β-catenin, leading to its nuclear translocation. Subsequently, nuclear β-catenin enhances the transcriptional activity of c-myc and cyclin D1 while suppressing the expression of RIPK3 and MLKL, thereby fostering BLCA progression and cisplatin resistance. Importantly, our clinical data suggest that the OTUB1/β-catenin/RIPK3/MLKL axis holds promise as a potential biomarker for BLCA.

Keywords: BLCA; OTUB1; chemoresistance; deubiquitination; β-catenin.

Figure 1

OTUB1 is elevated in BLCA and related to poor prognosis. A. Volcano plot of differentially expressed genes (DEGs) in TCGA BLCA data. B. The heatmap showed the expression of all members in the OTU deubiquitinase superfamily in BLCA and para-cancer tissues. C. Box plots of the relative mRNA expression levels of OTUB1 in BLCA and para-cancer samples from the TCGA database. D, E. The analysis of OTUB1 protein and mRNA expression in BLCA (T) and paired normal bladder (N) tissues by western blot and qRT- PCR. F. Representative immunohistochemical staining of OTUB1 and ki-67 in BLCA and paired normal bladder tissues (100X, 200X). G. Kaplan-Meier plots representing survival probabilities in 42 BLCA patients according to the relative expression level of OTUB1. Statistical analysis was conducted using the Student t-test and log-rank test.

Figure 2

Elevated OTUB1 is involved in the tumorigenesis of BLCA in vivo. Flow chart for the mouse model of spontaneous BLCA by feeding 0.1% BNN in water, these mice were treated with or without 0.1% BNN daily for 3 months. After stopping feeding BNN, all mice were divided into two groups according to time (1-6 months group and 7-12 months group). These bladders were removed and further handled for H&E staining and immunohistochemical staining (OTUB1 and ki-67). The left group from top to bottom: normal bladder structure, bladder carcinoma in situ, invasive BLCA; the Middle group from top to bottom: IHC staining for OTUB1 according to the sequence of H&E staining; Right group from top to bottom: IHC staining for ki-67 according to the sequence of H&E staining.

Figure 3

OTUB1 facilitates the proliferation and migration of BLCA. A. Relative OTUB1 expression in bladder epithelial immortalized cell SV-HUC and several BLCA cells by western blot and qRT-PCR. B, C. Relative OTUB1 expression following overexpressed or knockdown OTUB1 by western blot. D, F, G. MTT assay, clone formation experiments and healing assay showed the proliferation ability of BLCA cells following overexpressed or knockdown OTUB1. E. Transwell assay showed the invasion ability of BLCA cells following overexpressed or knockdown OTUB1. H, I, J. Cell cycle assay, clone formation experiments and healing assay showed the proliferation ability of BLCA cell after transfecting with shOTUB1 lentivirus. K. Relative cell cycle-related markers expression following overexpressed or knockdown OTUB1 by western blot.

Figure 4

OTUB1 interacts with β-catenin and regulates its stability. A. Heatmap of RNA-seq expression data for EJ cell transfected with control or shOTUB1 lentivirus. B. GSEA of RNA-seq data revealed that OTUB1 target genes were involved in Wnt/β-catenin signaling pathway. C, D. Relative expression of β-catenin following overexpressed or knockdown OTUB1. E. Relative expression of β-catenin following overexpressed or knockdown OTUB1 (treated with or without MG132 for 8h). F. Relative expression of β-catenin following overexpressed or knockdown OTUB1 (treated with MG132 or chloroquine for 8h). G. Relative expression of β-catenin following overexpressed or knockdown OTUB1 (treated with or without CHX for 0h, 4h, 8h and 24h). H. Relative mRNA expression of OTUB1 and β-catenin following knockdown OTUB1. I. The interaction between OTUB1 and β-catenin was determined by a co-immunoprecipitation assay.

Figure 5

OTUB1 maintains β-catenin stability depending on its deubiquitinase activity. A. Immunofluorescence assay showed the nuclear expression of β-catenin in BLCA cells transfected with OTUB1 plasmid. B. Relative expression of Wnt/β-catenin downstream targets following overexpressed or knockdown OTUB1. C. Schematic showed the functional domain and two mutant fragments in OTUB1. D, E. Immunoprecipitation assay showed that the 47-271 domain in OTUB1 interacts with β-catenin, further deubiquitinates and stabilizes β-catenin. F. Schematic showed the three amino acid mutant sites in OTUB1 (D88A; C91S; H265A; ASA: D88A, C91S and H265A). G, H. Immunoprecipitation assay showed that D88A and H265A in OTUB1 interact with β-catenin, further deubiquitinates and stabilize β-catenin; while C91S and ASA in OTUB1 do not. I. Immunoprecipitation assay showed that overexpressed OTUB1 restrains the ubiquitination of β-catenin, knockdown OTUB1 promotes the ubiquitination of β-catenin, while OTUB1 C91S has no effects. J. Schematic showed the functional domain and three mutant fragments in β-catenin. K. Immunoprecipitation assay showed that 1-694 domain fragment in β-catenin interacts with OTUB1. L. Immunoprecipitation assay showed that β-catenin serine33 mutation fails to interact with OTUB1.

Figure 6

OTUB1 promotes the progression of BLCA depending on β-catenin/RIPK3/MLKL/necroptosis signaling pathway. A. Relative expression of OTUB1, β-catenin and downstream targets following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; relative expression of OTUB1, β-catenin and downstream targets following knockdown OTUB1 and/or overexpressed β-catenin. B. The RNA expression of OTUB1, β-catenin, RIPK3 and MLKL in elevated OTUB1, β-catenin groups with or without XAV-939 treatment. C, E, F. MTT assay, clone formation experiments and healing assay showed the proliferation ability of BLCA cell following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; coupled with that following knockdown OTUB1 and/or overexpressed β-catenin. D. Transwell assay showed the invasion ability of BLCA cells following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; coupled with that following knockdown OTUB1 and/or overexpressed β-catenin.

Figure 7

OTUB1 is involved in cisplatin resistance of BLCA through β-catenin stabilization. A. Cell survival assay was determined in T24 control cell and T24 cisplatin-resistance cell. B. The changes in cell morphology between the T24 control cell and the T24 cisplatin-resistance cell. C. Relative expression of several chemoresistance markers (including MDR1, BCRP and YB-1) in T24 control cell and T24 cisplatin-resistance cell with or without cisplatin treatment. D. Relative expression of OTUB1 in T24 control cell and T24 cisplatin-resistance cell with or without cisplatin treatment. E. Relative expression of OTUB1, necroptosis-related markers (such as RIPK3, MLKL and P-MLKL), β-catenin and downstream proteins (including AXIN-2, C-myc, cyclin D1 and TCF1) in T24 cisplatin-resistance cells treated with gradient cisplatin concentration. F. Immunoprecipitation assay showed the relationship between OTUB1 and β-catenin in T24 cisplatin-resistance cells. G. Immunoprecipitation assay showed that gradient cisplatin concentration promotes the interaction between OTUB1 and β-catenin, and restrains the ubiquitination of β-catenin. H. Relative expression of OTUB1, necroptosis-related markers (such as RIPK3, MLKL and P-MLKL), β-catenin and downstream targets following elevated OTUB1, β-catenin with or without XAV-939 treatment in T24 cisplatin-resistance cell (control, OTUB1, OTUB1/XAV-939, β-catenin). I. Cell survival assay was determined in T24 cisplatin-resistance cells following overexpressed OTUB1 with or without 10uM XAV-939 (or knockdown OTUB1 with or without overexpressed β-catenin). J. Knockdown OTUB1 restrains the growth in T24 cisplatin-resistance mice bladder tumor in vivo.

Figure 8

Targeting the OTUB1/β-catenin axis suppresses the tumorigenesis and progression of BLCA in vivo. A. The flow chart to experimental design for the in vivo tumorigenesis assay in four different bladder cell types (control, OTUB1, OTUB1/XAV-939, β-catenin). B, C. The volume and growth of tumors in the following groups (control, OTUB1, OTUB1/XAV-939, β-catenin). D. Immunohistochemical staining showed the expression of OTUB1, ki-67 and β-catenin in mice tumors among control, OTUB1, OTUB1/XAV-939 and β-catenin groups. E. Relative expression of OTUB1, necroptosis-related markers (such as RIPK3, MLKL and P-MLKL), β-catenin and downstream targets in mice tumors among control, OTUB1, OTUB1/XAV-939 and β-catenin groups. F. Simplified schematic to a working model for regulation of β-catenin stability and cancer tumorigenesis and chemoresistance by OTUB1.