BAP1-mediated stabilization of SUFU enhances E2F1 activity and promotes colorectal tumorigenesis

Abstract

Background: The Hedgehog (Hh) signaling pathway plays a pivotal role in tumorigenesis. SUFU (Suppressor of Fused), a key negative regulator of Hh signaling, also interacts with additional signaling pathways involved in cancer progression. However, its specific function and regulatory mechanisms in colorectal cancer (CRC) remain poorly understood.

Methods: Using Co-immunoprecipitation (Co-IP), we identified BAP1 as a candidate deubiquitinating enzyme (DUB) that interacts with SUFU. Western blotting was used to investigate the molecular mechanism by which BAP1 regulates SUFU protein stability. Integration of public databases and immunohistochemical (IHC) staining of CRC patient samples revealed altered expression both BAP1 and SUFU in CRC. Functional assays, including CCK-8 cell proliferation assays and xenograft tumor models, were conducted to evaluate the biological impact of BAP1-SUFU signaling. Furthermore, transcriptomic analyses and rescue experiments were performed to investigate the downstream regulatory mechanisms of the BAP1-SUFU axis in CRC cell proliferation.

Results: We demonstrated that BAP1 regulates the ubiquitination and protein stability of SUFU via its UCH enzymatic domain. Analysis of CRC clinical samples and public datasets revealed that BAP1 and SUFU are concurrently upregulated in tumor tissues. Functional experiments in vivo confirmed that BAP1 and SUFU promote CRC cell proliferation and clonogenic potential through modulation of the cell cycle. Pharmacological inhibition of BAP1 resulted in reduced SUFU protein levels and significantly suppressed CRC cell proliferation. Rescue experiments further showed that overexpression of SUFU partially restored cell proliferation in the presence of BAP1 inhibition, suggesting a functional dependency. Transcriptomic and mechanistic analyses revealed that disruption of the BAP1-SUFU axis alters cell-cycle transcriptional programs through a Hh-independent mechanism and implicates E2F1 as a downstream regulator, which was confirmed by Western blot and rescue experiments demonstrating its essential role in driving the proliferative phenotype. Moreover, knockdown of BAP1 or SUFU, as well as pharmacological inhibition of BAP1, significantly suppressed tumor growth in xenograft models and reduced E2F1 protein levels, further elucidating the mechanistic basis of this regulatory axis and demonstrating its therapeutic potential in vivo.

Conclusion: These findings highlight the critical role of SUFU in promoting CRC cell proliferation through its regulation by BAP1. Targeting BAP1 enzymatic activity offers a promising therapeutic strategy to modulate SUFU levels and suppress tumor growth. The development of specific BAP1 inhibitors may represent a novel and effective approach to improve outcomes in CRC.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12964-025-02627-9.

Keywords: BAP1; Colorectal cancer; Deubiquitinase inhibitors; E2F1; SUFU.

Fig. 1

Screening of the UCH family identifies BAP1-SUFU interaction and positive regulation of SUFU. A Co-immunoprecipitation (Co-IP) screening was conducted to identify protein-protein interactions between overexpressed ubiquitin carboxyl-terminal hydrolase (UCH) family members with SUFU in HEK293T cells. B Immunofluorescence analysis of HEK293T cells co-expressing BAP1 and SUFU, visualized by confocal microscopy. Bar = 10 μm. C Western blot analysis was employed to assess endogenous SUFU protein levels following 48 h of BAP1 overexpression in HEK293T cells. D qPCR analysis was performed to measure endogenous SUFU mRNA levels following 48 h of BAP1 overexpression at varying doses in HEK293T cells (n = 3). Data are presented as mean ± SEM. ns, not significant; **** p < 0.0001. E CHX chase assay was performed to investigate the impact of BAP1 overexpression on the degradation rate of endogenous SUFU protein in HEK293T cells. After 48 h of BAP1 overexpression, cells were treated with CHX, and SUFU protein levels were monitored at 0, 2, 4, 6, 8, and 10 h to assess protein stability. F Quantification of SUFU and GAPDH band intensities from Western blots. E was conducted using ImageJ software. The resulting SUFU/GAPDH ratios were plotted over time to generate SUFU degradation curves (n = 3). Data are presented as mean ± SEM; ** p < 0.01. G Western blot was used to assess SUFU levels after BAP1 knockdown in HEK293T, with MG132 confirming proteasome involvement (H, I and J) SUFU was immunoprecipitated from HEK293T cells with BAP1 overexpression (H), knockdown (I) or BAP1 inhibitor treatment. J to assess changes in SUFU ubiquitination levels. K A schematic diagram illustrates the design of BAP1 domain deletion mutants, constructed to investigate which regions are essential for mediating protein interactions L Co-IP screening of BAP1 truncation mutants with SUFU was performed in HEK293T cells to identify the domains mediating their interaction, with results validated by Western blot analysis

Fig. 2

BAP1 and SUFU contribute to colorectal cancer development. A Expression profiles of SUFU and BAP1 in colorectal cancer (CRC) tissues versus normal controls were analyzed using datasets from GSE37182, and GSE39582. Data are expressed as mean ± SD, **** p < 0.0001. B Detection of BAP1 and SUFU protein levels in CRC patient tissues by Western blot analysis. n = 4. C and D Immunohistochemical (IHC) staining was performed on paraffin-embedded sections from CRC patients (n = 3) to evaluate SUFU and BAP1 expression in tumor versus adjacent non-tumor tissues. Bar = 50 μm. E and F Kaplan-Meier survival analysis, using data from the Kaplan-Meier Plotter website, was used to assess the association between BAP1 and SUFU expression levels and relapse-free survival (RFS) as well as overall survival (OS) in CRC patients

Fig. 3

BAP1 modulates colorectal cancer cell proliferation via SUFU.A Western blot analysis was conducted to verify the knockdown efficiency of BAP1 and SUFU following siRNA-mediated silencing in HCT116 and SW480 cells at 72 h post-transfection. B Proliferation curves of HCT116 and SW480 colorectal cancer cells were generated using the CCK-8 assay following BAP1 or SUFU knockdown (n = 6). Data are presented as mean ± SEM, **** p < 0.0001. C Colony formation assays were performed to evaluate the proliferative capacity of HCT116 and SW480 cells following BAP1 or SUFU knockdown. Cells were seeded in 6-well plates and cultured for 10 days. D Quantification of colony numbers was conducted using ImageJ, with colonies defined as clusters exceeding (n = 6). Data are expressed as mean ± SEM, *** p < 0.001, **** p < 0.0001. E and F Flow cytometry analysis was employed to assess cell cycle distribution in HCT116 and SW480 cells after BAP1 or SUFU knockdown, with particular attention to the proportion of cells in the G0/G1 phase. G Cell cycle distribution analyzed using ModFit LT software following BAP1 or SUFU Knock down, with statistical summary of G0/G1, S, and G2/M phase proportions, Data are mean ± SEM, n = 3, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. H Western blot analysis was used to confirm SUFU overexpression efficiency in HCT116 and SW480 cells. The higher molecular weight band represents the exogenously expressed SUFU-Myc fusion protein introduced via the pCDH vector. I Proliferation curves of HCT116 and SW480 cells following SUFU overexpression were measured using the CCK-8 assay (n = 6). Data are expressed as mean ± SEM, **** p < 0.0001. J Proliferation curves of HCT116 cells with SUFU re-expression following BAP1 knockdown were determined using the CCK-8 assay after siRNA-mediated silencing (n = 6). Data are expressed as mean ± SEM, **** p < 0.0001. K Endogenous SUFU protein levels in HCT116 and SW480 cells were evaluated by Western blot analysis following treatment with increasing concentrations of BAP1 inhibitor. L Proliferation curves of HCT116 and SW480 cells treated with varying concentrations of BAP1 inhibitor were generated using the CCK-8 assay (n = 6). Data are expressed as mean ± SEM, ns, not significant, ** p < 0.01, **** p < 0.0001. M Colony formation assays were performed to evaluate the proliferative capacity of HCT116 and SW480 cells treated with varying concentrations of specify BAP1 inhibitor. Cells were seeded in 6-well plates and cultured for 10 days. N Colony formation assays were conducted in HCT116 and SW480 cells treated with varying concentrations of specify BAP1 inhibitor (n = 6). Data are expressed as mean ± SEM. ns, not significant, * p < 0.05, ***p < 0.001, **** p < 0.0001. O Proliferation curves of HCT116 cells overexpressing SUFU and treated with varying concentrations of BAP1 inhibitor were measured using the CCK-8 assay (n = 6). Data are expressed as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01

Fig. 4

The BAP1-SUFU axis regulates E2F1 levels to influence cell proliferation. A Overview of the transcriptomic analysis workflow. B Volcano plots illustrate differentially expressed genes (DEGs) in HCT116 colorectal cancer cells following BAP1 or SUFU knockdown, identified using RNA sequencing. DEGs were defined by a threshold of log₂ (fold change) > 2 and a false discovery rate (FDR) < 0.05. C Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the DEGs to identify significantly altered biological pathways. D Overlapping genes altered by both BAP1 and SUFU knockdown were identified through comparative analysis of RNA sequencing datasets. E KEGG functional enrichment analysis was conducted on the 272 overlapping genes identified in (C) to determine significantly enriched biological pathways. F A volcano plot depicts DEGs in HCT116 cells following SUFU re-expression following BAP1 knockdown. DEGs were identified based on thresholds of DEGs defined by log₂ (fold change) > 2 and FDR < 0.05. G KEGG functional enrichment analysis was performed on the DEGs identified in (E) to elucidate pathways the biological pathways modulated by SUFU re-expression following BAP1 knockdown. H A heatmap displays the normalized FPKM values for genes associated with cell cycle, proliferation, and apoptosis, based on RNA sequencing data. I Gene Set Enrichment Analysis (GSEA) was conducted to identify significantly enriched signaling pathways linked to gene expression changes following BAP1 knockdown, SUFU knockdown, and SUFU reconstitution in BAP1-depleted HCT116 cells. J RNA sequencing analysis was performed to investigate the expression profiles of E2F1-regulated genes in HCT116 cells following BAP1 or SUFU knockdown. K Positive correlation between BAP1 expression and E2F1 mRNA levels, along with a set of E2F1 target genes, in TCGA CRC-cancer cohorts. L Western blot analysis was used to measure E2F1 protein levels in HCT116 cells following BAP1 or SUFU knockdown. M Western blot analysis was conducted to assess E2F1 protein levels in HCT116 cells upon SUFU re-expression in the context of BAP1 knockdown. N qPCR analysis was performed to measure endogenous E2F1 mRNA levels following in SUFU-knockdown, as well as SUFU-knockdown with E2F1 restoration conditions, Data are expressed as mean ± SEM, n = 3, **** p < 0.0001. O Cell proliferation assay showing that SUFU knockdown significantly reduces cell proliferation, whereas restoration of E2F1 expression rescues the proliferative capacity. Data are mean ± SEM, n = 3, ****p < 0.0001

Fig. 5

BAP1 and SUFU knockdown suppresses tumor growth. A Growth curve of xenograft tumors in nude mice implanted with stable cell lines harboring BAP1 or SUFU knockdown (n = 9). Data are expressed as mean ± SEM; **** p < 0.0001. B Representative images of xenograft tumors excised from nude mice at the experimental endpoint. C Quantitative analysis of tumor weights (n = 9). Data are expressed as mean ± SEM. **** p < 0.0001. D Western blot analysis of SUFU and BAP1 protein levels in xenograft tumor tissues to evaluate knockdown efficiency. E IHC staining for Ki-67 in tumor sections to assess cell proliferation. Bar = 50 μm. F TUNEL staining of tumor sections to detect apoptotic cells. Bar = 50 μm. G and H Quantification of Ki-67 and TUNEL-positive areas using ImageJ (n = 3). Data are expressed as mean ± SEM, **** p < 0.0001. I Representative images of HCT116 xenograft tumors in nude mice treated with BAP1 inhibitor (50 mg/kg body weight, administered every other day) to evaluate therapeutic efficacy. J Tumor weight of excised xenografts following BAP1 inhibitor treatment (n = 7). Data are expressed as mean ± SEM, **** p < 0.0001. K IHC staining of E2F1 in tumors from control, BAP1-knockdown, SUFU-knockdown, and BAP1 inhibitor-treated groups. Bar = 100 μm

Fig. 6

Proposed model of the BAP1-SUFU-E2F1 axis in colorectal cancer and its therapeutic potential