STAMBP-Mediated K63 Deubiquitination of E2F1 Release E2F1 from RB Repressive Complex to Drive Bladder Cancer Progression

STAMBP-Mediated K63 Deubiquitination of E2F1 Release E2F1 from RB Repressive Complex to Drive Bladder Cancer Progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article STAMBP-Mediated K63 Deubiquitination of E2F1 Release E2F1 from RB Repressive Complex to Drive Bladder Cancer Progression Shangze Li, Tao Liu, Qipeng Shu, Li Yu, Chun-Mei Jiang, Huangheng Tao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8327775/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The canonical RB1-E2F regulatory model depends on RB phosphorylation-induced allosteric changes during the cell cycle. However, extensive RB mutations across cancers indicate the existence of phosphorylation-independent mechanisms governing RB–E2F complex stability. Here, we report a novel regulatory axis where enhanced E2F1 activity inversely correlates with K63-linked ubiquitination levels, independent of changes in RB1 phosphorylation status. Through systematic deubiquitinase profiling, we identify STAM Binding Protein (STAMBP)—a K63-specific deubiquitinase overexpressed in bladder tumors and correlated with advanced disease and poor survival—as the key enzymatic regulator. Mechanistically, STAMBP binds E2F1 and removes K63 chains at lysines 161/164, destabilizing the RB1-E2F1 repressive complex while maintaining RB1 phosphorylation homeostasis. This enhances E2F1 transcriptional activity, driving cell cycle target gene expression and promoting malignant progression through proliferation and invasion. Genetic loss of STAMBP suppresses tumor growth in vitro and in vivo. Bladder-specific Stambp knockout delays carcinogen-induced tumor progression and improves survival, while pharmacological inhibition with BC1471 selectively blocks proliferation in STAMBP-high cells without toxicity. Together, these findings establish a 'Dual-Lock' paradigm: K63-linked ubiquitin chains act as a molecular scaffold stabilizing the RB1-E2F1-HDAC1 complex, whereas STAMBP-mediated deubiquitination triggers oncogenic E2F1 activation. This work nominates STAMBP as a biomarker-driven therapeutic target for precision oncology in bladder cancer. Biological sciences/Cancer/Oncogenes Health sciences/Diseases/Urogenital diseases STAMBP E2F1 deubiquitination bladder cancer RB1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bladder cancer (BC) is the most common malignant tumor of the urinary system and is projected to cause 17,420 deaths among 84,870 new cases in the United States in 2025( 1 ). Globally, BC exhibits a pronounced gender disparity, with incidence rates in men being two- to four-fold higher than in women( 2 ). The therapeutic strategy has rapidly evolved from surgery-chemotherapy combinations to precision approaches that incorporate molecularly targeted agents(e.g., Erdafitinib for FGFR3/2-altered tumors), immune checkpoint inhibitors (e.g., pembrolizumab ), and antibody-drug conjugates( 3 ). Notably, pembrolizumab significantly improved median overall survival to 16 months compared with 9 months for chemotherapy in cisplatin-ineligible patients( 4 ), while Erdafitinib extended survival to 12.1 months versus 7.8 months in chemotherapy-treated controls (HR = 0.64; 95% CI: 0.47–0.88)( 5 ). Critically, actionable targets such as FGFR alterations are present in only 15–20% of patients( 6 ), leaving > 80% without molecularly guided therapies. Consequently, further research into the molecular mechanisms of BC and the identification of broader therapeutic targets are urgently needed to improve patient outcomes. E2F transcription factor 1 (E2F1), a master regulator of cell cycle-dependent transcription, governs diverse cellular processes including apoptosis, DNA damage repair, and metabolism( 7 ). Dysregulation of E2F1 has been observed in multiple cancer types( 8 , 9 ). Notably, E2F1 expression is particularly critical for progression from superficial to invasive disease. Molecular subtyping reveals that T1-luminal genomically unstable tumors exhibit significant enrichment of E2F1 transcription factor motifs ( 10 ). Overexpression of E2F1 drives the transition from superficial to invasive stages by activating cell cycle targets, such as CDK1 and CCNE1, through promoter motif enrichment( 11 ). In addition, E2F1 activity is critically regulated by direct binding of the retinoblastoma protein (RB1). During G1 phase, hypo-phosphorylated RB1 sequesters E2F1 in a repressive complex, inhibiting transcriptional activation of S-phase genes until CDK-mediated RB1 phosphorylation result in a conformational change to release E2F1. This release allows E2F1 to function as an active transcription factor that promotes the expression of genes essential for entering the S phase( 12 , 13 ). E2F1’s transcriptional activity can be modulated by its association with co-activators such as histone acetyltransferases (HATs) and co-repressors such as histone deacetylases (HDACs)( 14 ). Beyond this canonical model, tumors frequently exhibit continuous E2F1 activation independent of RB1 phosphorylation, suggesting an alternative RB1 conformation–independent regulatory mechanism. The emerging research highlights the critical role of post-translational modifications (PTMs) in modulating E2F1 stability and activity.​ For instance, ubiquitination pathways significantly regulate E2F1 proteostasis: APC/C Cdh1 mediates K11-linked degradation, whereas POH1 stabilizes E2F1, promoting survival signals like Survivin and FOXM1 during tumorigenesis( 15 , 16 ). However, current studies focus predominantly on PTM-mediated effects on E2F1 stability or transcriptional activity, without addressing potential crosstalk with the RB1-E2F1 complex dynamics.​​ Specifically, whether and how specific PTMs directly disrupt or reinforce the RB1-E2F1 interaction remains unexplored, highlighting a critical gap in understanding PTM-driven regulation of this core cell cycle machinery. Protein homeostasis is dynamically regulated by PTMs, especially ubiquitination, which involves E1–E2–E3 ligase–mediated substrate tagging and removal of ubiquitin chains by deubiquitinating enzymes (DUBs). Dysregulation of these processes significantly contributes to oncogenesis( 17 ). The ubiquitin molecule can form connections through seven distinct lysine sites (K6, K11, K27, K29, K33, K48, and K63)( 18 ). Each conferring distinct functional consequences. K63-linked ubiquitination can influence various protein characteristics, including protein interactions, translocation, and activation processes( 19 ). For example, TRAF2 K63-linked ubiquitination mediates interactions with TAB2/3 and activates the downstream kinases IKK and JNK( 20 ). K63-linked ubiquitination of E2F1 has also been reported. The E3 ubiquitin ligase cIAP1 catalyzes K63-linked ubiquitination of E2F1 at lysines 161 and 164 (K161/K164). These specific residues are essential for E2F1-mediated gene activation( 21 ). Lin et al. identified UCH37 as the first DUB that directly regulates E2F1 activity. They proposed the following model: E2F1 is ubiquitinated with K63-linked ubiquitin chains, and this modification represses its transcriptional activity. UCH37 alleviates this repression by removing K63-linked ubiquitin chains from E2F1( 22 ). The molecular basis of why K63-linked ubiquitination suppresses E2F1 activity, however, remains unresolved. The STAM-binding protein (STAMBP), also known as AMSH (associated molecule with the SH3 domain of STAM), is a zinc-dependent metalloprotease that belongs to the JAMM/MPN family of DUBs. This enzyme specifically hydrolyzes K63-linked polyubiquitin chains, thereby modulating endosomal sorting and signal transduction pathways( 23 ). Recent studies have shown that STAMBP is closely related to the occurrence and development of many diseases, and STAMBP has an important role in the field of oncology. In triple-negative breast cancer, high levels of STAMBP were correlated with poor prognosis. STAMBP stabilized the RAI14 protein by suppressing the K48-linked ubiquitination of RAI14, thereby preventing its proteasomal degradation( 24 ). In lung adenocarcinoma, it regulates EGFR ubiquitination to activate pro-EMT signaling( 25 ). Despite these findings, the pathological role of STAMBP in bladder cancer remains entirely unknown. In the present study, we integrated multi-platform analyses and identified significant upregulation of STAMBP in bladder tumors. Elevated STAMBP expression was associated with reduced disease-free survival, supporting its prognostic relevance. The pathological overexpression of STAMBP in malignant epithelia was confirmed by immunohistochemical validation in clinical cohorts. Functionally, we demonstrate that STAMBP interacts with E2F1 and selectively suppresses K63-linked polyubiquitination to enhance E2F1 transcriptional activity. Crucially, K63-linked ubiquitin chains serve as molecular scaffolds that stabilize the RB1-E2F1 complex, and their deubiquitination triggers dissociation of the repressor-transcription factor complex, liberating transcriptionally active E2F1. Based on these findings, we propose a 'Dual-Lock' mode. In this model, RB1-E2F1 binding affinity is co-regulated by two mechanisms: the canonical RB1 pocket conformation acting as a gatekeeper, and K63-polyUb chains functioning as allosteric stabilizers at the protein-protein interface. This model addresses the long-standing question of constitutive E2F1 activation in cancers with intact RB1 and establishes K63-linked ubiquitination as a critical rheostat of cell cycle control. Materials and Methods Cell Culture and Cell Lines Cell lines including T24 and HEK293T cell lines were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (CASS). HEK293T cells were cultured in DMEM medium and T24 cells were cultured in McCoy's 5A medium. All culture medium were supplemented with 10% fetal bovine serum (FBS; Gibco, China), 100 U/ml penicillin-G sodium, and 100 mg/ml streptomycin sulfate. All cells were cultured in an incubator maintained at 37°C with 5% CO₂. Reagents and Antibodies GAPDH (Cat. #60004-1-Ig) were purchased from Proteintech; FLAG (Cat. #M185-6), MYC (Cat. #M047-3), HA (Cat. #M180-3) were purchased from MBL (Medical & Biological Laboratories Co., Ltd.); Antibodies against STAMBP (Cat. #5245) and E2F1 (Cat. #3742) for immunoprecipitation (IP) were obtained from Cell Signaling Technology; Antibodies against STAMBP (Cat. #A7065), E2F1 (Cat. #A16720), CDC2 (Cat. #28439) for immunohistochemistry (IHC) were purchased from Abclonal (Abclonal, United States); Antibodies against DHFR (Cat. #43497) for immunohistochemistry (IHC) were purchased from Cell Signaling Technology; BC1471(HY-122883) were purchased from Medchemexpress. Tissue microarrays was purchased from Shanghai Outdo Biotech Company (Cat. HBlaU066Su01). Genetic Knock-Out of STAMBP in T24 Cells The STAMBP gene knockout in T24 cells was conducted utilizing the CRISPR-Cas9 technology. In summary, we designed sgRNAs for STAMBP using the online tool available at http://crispor.tefor.net/ . Subsequently, the sgRNA was ligated into the lentiCRISPRv2 vector (Addgene, USA). The resulting vector containing the gRNA was co-transfected with packaging plasmids into HEK293T cells to facilitate virus packaging. The supernatant, which contained viral particles, was collected and utilized to infect T24 cells. Following 48 hours of infection, positive cells were selected through puromycin (1 µg/ml) treatment. Approximately 5 days later, the positive clones were distributed into 96-well plates using a gradient dilution method. After 14 days, single clones were picked. A portion of the screened monoclonal cells was utilized for protein extraction to conduct western blotting experiments aimed at verifying the successful knockout of STMBP. The sgRNA sequences: sgRNA-F: 5′-caccGGATAATCTCAACTCCAGAG-3′, sgRNA-R e: 5′-aaacCTCTGGAGTTGAGATTATCC-3′. Plasmids and sgRNA Flag-STAMBP, Flag-STAMBP D348A HA-HDAC1, Myc-RB1, HA-E2F1, Myc-E2F1, HA-E2F1 K161/164R . To overexpress STAMBP, we purchased the overexpression plasmids for STAMBP from Miao Ling Biotech’s plasmid platform. The sgRNA sequences: sgRNA-F, 5'-caccGGATAATCTCAACTCCAGAG-3'; sgRNA-R, 5'-aaacCTCTGGAGTTGAGATTATCC-3'. CCK8 assays Cells were seeded in a 96-well plate at a density of 2000 cells per well. Testing was conducted every 24 hours over a period of 4 days. At each time point, 10 µl of CCK8 (Biosharp, Wuhan, China) was mixed with 100 µl of culture medium and added to the test wells. The plate was subsequently incubated at 37°C for 2 hours, and the absorbance was measured at OD450 nm using a microplate reader. Colony formation assays Cells were seeded in a 6-well plate at a density of 1200 cells per well. The plates were incubated at 37°C for 10 days. Then the colonies were fixed using 4% paraformaldehyde for 30 minutes and stained with a 0.2% crystal violet solution for an additional 30 minutes. The clones were photographed and counted. Transwell Migration Assays 500 µl culture medium containing 20% FBS was added to the lower chamber. Serum-free medium was placed in the upper chamber. Cells were seeded in the upper chamber. After 24 hours of incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Photographs were taken, and the cells were counted. RNA extraction and real-time PCR Trizol reagent (Ambion, USA) was used to isolate total RNA. The ABScript II RT Master Mix (Tesingke, Wuhan, China) was used to synthesized cDNA following the manufacturer’s protocol. The real-time PCR analysis was conducted on a Bio-Rad CFX96 instrument (Hercules, CA, USA) and the quantification was performed utilizing GraphPad Prism 8. The primer sequences used are listed in Table S1. Western blotting Cells were collected and lysed using RIPA buffer containing Phosphorylase Inhibitors (Biosharp, Wuhan, China) and Protease Inhibitors (Biosharp, Wuhan, China).The total proteins form each group were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Shanghai, China). After treatment with 5% skim milk for 1 hour at room temperature, the PVDF membrane was incubated with the primary antibody overnight at 4°C. Following three washes with TBST, the membrane was incubated with secondary antibody for 2 hours at room temperature and washed with TBST. The protein expressions on the PVDF membrane were detected using enhanced chemiluminescence (Biosharp, Wuhan, China). Tissue Microarray and Immunohistochemistry The Tissue Microarray (Outdo Biotech, Shanghai, China) comprises of 116 bladder cancer tissues and 46 adjacent non-cancerous tissues. The slides were treated with a 10% solution of H 2 O 2 and subsequently permeabilized using 0.1% TX-100. Following the reagent supplier's protocol. the primary and secondary antibodies were applied to the slides for incubation. The DAB protein coloring solution was then used to stain the protein expression, which was observed under a microscope. The Histoscore was calculated by multiplying the staining intensity score by the positivity rate observed in each tissue sample. Luciferase reporter assay 5 ng of the pRL-CMV plasmid, 300 ng of E2F-Luc vectors (Yeasen, China) and appropriate amount of Flag-STAMBP plasmid or an empty vector were transfected into T24 cells or HEK293T cells. After 48 hours, luciferase activity was measured using a dual-luciferase assay kit (Promega), with Promega luciferase system. Coimmunoprecipitation (Co‑IP) assay Cells were lysed in ice-cold lysis buffer (30 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The resulting lysates were subjected to centrifugation at 12,000 rpm for 10 minutes at 4°C, the cell debris was discarded, and the cell supernatant was retained. G-agarose beads (Smart-Lifesciences, Changzhou, China) precoated with the indicated antibodies were added to the supernatant with lysed cells, which were incubated at 4°C. Six hours later, the Co-IP proteins were subjected to Western blot analysis. Deubiquitination analysis HEK293T cells were transfected with indicated plasmids, and 24 hours post-transfection, the cell culture medium was replaced with a fresh culture medium containing10 µM of MG132. At 36 hours post-transfection, the cells were washed using PBS and lysed with SDS lysis buffer (10% SDS in PBS). The lysates were heated to 95°C, followed by the addition of a twofold volume of modified RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and protease inhibitor). Subsequently, the lysates were cooled on ice for 30 min and centrifuged at 12,000×g for 15 min at 4°C. Finally, the supernatant was subjected to anti-HA immunoprecipitation and Western blot analysis. HE staining The prepared tissue sections were deparaffinized through consecutive treatments with xylene and varying concentrations of alcohol, with each immersion lasting 5 minutes. Mayer's hematoxylin (Wako, Japan) was utilized for nuclear counterstaining, which was subsequently followed by treatment with a 1% eosin solution for additional counterstaining. Before being observed under an Olympus microscope (Tokyo, Japan), the sections were cleared and dehydrated with the use of alcohol. Animal study Tumor Xenografts: Four-week-old nude mice were purchased from the Experimental Animal Center of Wuhan University (Wuhan, China). Following a week of acclimatization, the mice were subcutaneously injected with 4 × 10 6 cells. Tumors were measured every other day after the appearance of subcutaneous tumors. The volume of the tumors was determined using the formula: volume = (length×width 2 ) × 0.5. After 31 days of observation, the nude mice were euthanized using CO 2 asphyxiation, and the transplanted tumors were taken out in a biosafety cabinet. Each group of tumors was imaged, and both their volume and weight were quantified. The tumor tissues were fixed with 4% paraformaldehyde. and subsequent staining was carried out using either HE or IHC techniques. AOM/DSS-induced colorectal cancer model: BALB/c mice aged 5–6 weeks were obtained from the Experimental Animal Center of Wuhan University (Wuhan, China). Upk2-Cre and Stambp flox/flox mice were procured from the Model Animal Research Center of Nanjing University. Upk2-Cre; Stambp flox/flox and Stambp flox/flox male mice were generated through selective breeding. The mice (5 weeks old) were administered tamoxifen at a dosage of 75 mg/kg for 5 days. Three weeks later, the mice were provided with drinking water with or without 0.05% BBN. Mouse weights were monitored every 3 days. Mice were sacrificed in the 26th week, and both tumor weights and volumes were recorded. Additionally, all mice underwent pathological examination. The Survival percentage rate of tamoxifen-treated mice was also evaluated. All mice were maintained under SPF conditions with a 12:12 h dark: light cycle. All efforts were taken to reduce the suffering of the animals. Database analysis The TIMER, GEPIA, and UALCAN databases were employed to analyze the expression of STAMBP and assess its prognostic significance. The clinical relevance of STAMBP in bladder cancer patients was evaluated using the Sanchez-Carbayo Bladder 2 and Dyrskjot Bladder 3 datasets. These datasets were sourced from Oncomine ( https://www.oncomine.org ). We applied the log-rank test and Cox regression analyses to generate Kaplan–Meier curves. Statistical analysis The statistical significance of the entire dataset was assessed using GraphPad Prism software. The mean ± SEM values were used to present all data. Student's t -tests or one-way ANOVA were conducted for statistical analysis, considering P < 0.05 as the threshold for determining statistical significance. Results Amplification of STAMBP was associated with worse prognosis To examine the expression of STAMBP in bladder cancer, we analyzed datasets from GEPIA, UALCAN, and the Sanchez-Carbayo and Dyrskjot cohorts. The results revealed that STAMBP expression was significantly higher in bladder cancer tissues than in normal bladder tissues ( P < 0.05) (Fig. 1 A-D). Further analysis of the GEPIA database showed a negative correlation between high STAMBP expression and disease-free survival in patients with bladder cancer (Fig. 1 E), suggesting that elevated STAMBP may serve as an independent prognostic marker of poor outcomes. To validate these findings, immunohistochemical assessments was performed using tissue microarrays comprising 56 bladder cancer tissues and 10 normal bladder tissues. The results from the tissue microarray analysis indicated that STAMBP expression was significantly higher in bladder cancer tissues than in adjacent normal paracancerous tissues (Fig. 1 F, G). Collectively, these results highlight the potential oncogenic role of STAMBP in the development and progression of bladder cancer. Overexpression and knockout of STAMBP affect the proliferation, migration, and invasiveness of bladder cancer cell lines. To explore the function of STAMBP in bladder cancer cells, T24 cells were transfected with either a FLAG-STAMBP (wild-type) plasmid or a FLAG-STAMBP D348A -a catalytic-inactive mutant( 23 ). The expression levels of STAMBP were subsequently evaluated by western blotting (Fig. 2 A). Moreover, T24 cell lines lacking STAMBP (KO1/KO2) were created using the CRISPR/Cas9 approach, and the absence of STAMBP expression was validated by western blotting (Fig. 2 B). To assess cell viability and tumorigenic potential, both colony formation and cell growth assays were performed. The results showed that the ectopic expression of STAMBP enhanced colony formation and increased viability of T24 cells in comparison to the parental T24 cells. Conversely, transfection with STAMBP D348A had no effect on either colony formation or the proliferation (Fig. 2 C, E). Consistently, wild-type (WT) T24 cells displayed robust colony-forming capacity, whereas the STAMBP-knockout T24 cells generated considerably fewer colonies (Fig. 2 D, F). Furthermore, Transwell assays further revealed that STAMBP overexpression enhanced cell migration, whereas the mutation at the deubiquitination-catalyzing site in STAMBP did not alter migratory ability (Fig. 2 G). Conversely, STAMBP deficiency inhibited the migration of T24 cells (Fig. 2 H). Collectively, these findings demonstrate that loss of STAMBP adversely affects the proliferation, viability, and migration of T24 cells. STAMBP activates the E2F signaling pathway To investigate the how STAMBP downregulation affects bladder cancer cell proliferation and migration, we performed RNA-Seq analysis using T24 and STAMBP-knockout T24 cells. KEGG pathway enrichment analysis was conducted to revealed that the cell cycle pathway was significantly enriched in T24 cells (Fig. 3 A). Gene Set Enrichment Analysis (GSEA) results indicated that the cell cycle and E2F signaling pathways were significantly enriched in T24 cells (Fig. 3 B). Furthermore, we analyzed bladder cancer samples from the TCGA database, dividing patients into high and low expression groups based on STAMBP expression levels. Then, we conducted GSEA analysis, which corroborated the cellular findings, demonstrating significant enrichment of the cell cycle and E2F signaling pathways in bladder cancer tissues with high STAMBP expression (Fig. 3 C). To verify this result, we performed a luciferase signaling pathway assay in HEK293T cells to determine whether STAMBP could activate the relevant signaling pathways. The findings indicated that STAMBP can activate several signaling pathways, with the E2F1 pathway exhibiting the strongest and dose-dependent activation (Fig. 3 D, E). We analyzed the relationship between STAMBP and cell cycle and E2F-targeted genes using the cBioPortal database, which revealed certain correlations between STAMBP and the genes CDCA2, DHFR, and PCNA (Fig. 3 F). To validate these findings, we employed real-time quantitative PCR and Western blotting techniques. STAMBP knockout markedly reduced mRNA levels of cell cycle and E2F-targeted genes (CDC2, CCNA, CCND3, DHFR, E2F2, PCNA, and RNAGAP) (Fig. 3 G). Conversely, in T24 cells overexpressing STAMBP, there was a marked increase in the mRNA levels of these genes (Fig. 3 H). Additionally, Western blotting results demonstrated that the absence of STAMBP resulted in decreased protein levels of CDC2 and DHFR (Fig. 3 I), and increased expression of both proteins under STAMBP overexpression (Fig. 3 J). Together, these results demonstrate that STAMBP positively regulates the cell cycle and E2F signaling pathways. STAMBP dismantles the RB1-E2F1 complex via K63 deubiquitination of E2F1 To further explore the mechanism by which STAMBP activates the E2F pathway, we first tested the phosphorylation level of RB1, the result revealed that STAMBP does not alter RB1 phosphorylation status (Fig. 3 J), indicating a phosphorylation-independent regulatory mechanism. Given that STAMBP is a K63 specific deubiquitinase and according to previous study, we hypothesis that STAMBP enhances the transcriptional activity by releasing it from RB1 through cleavage of the K63-linked ubiquitin chain. Firstly, the Co-IP experiments were performed to test the connection between E2F1 complex and STAMBP. The results demonstrated that STAMBP interacted with E2F1, RB1, and HDAC1 (Fig. 4 A-C). Notably, E2F1 exhibited particularly strong binding with STAMBP (Fig. 4 A). Endogenous Co-IP experiments in T24 cells further confirmed that STAMBP interacts with E2F1 (Fig. 4 D). Subsequently, we aimed to investigate whether the ubiquitination level of E2F1 is regulated by STAMBP. The results demonstrated that STAMBP significantly reduced the ubiquitination of E2F1, while the STAMBP D348A had no effect, indicating that E2F1 ubiquitination depends on STAMBP deubiquitinating activity (Fig. 4 E). To determine whether K63-linked polyubiquitination is specifically removed from E2F1 by STAMBP, we employed an E2F1K161/164R double mutant that blocks. This mutant blocks K63-linked polyubiquitination at known regulatory sites critical for E2F1 stability( 21 ). As shown in Fig. 4 F, STAMBP expression reduced K63-linked ubiquitination in wild-type E2F1, whereas its inhibitory effect was ​​significantly reduced​​ on the mutant. Additionally, this site-specific regulation was further validated using a K63-only ubiquitin mutant (K63O), which forms exclusively K63-linked chains. The results indicated that in the presence of STAMBP, the K63 ubiquitination level of E2F1 was significantly reduced; however, while ubiquitination of the E2F1 K161/164R mutant remained unchanged (Fig. 4 G). These results indicate that STAMBP decreases K63-linked ubiquitination of E2F1 by targeting lysine residues 161 and 164. To investigate whether STAMBP-mediated removal of K63-linked ubiquitin from E2F1 affects its interaction with RB1, we conducted Co-IP. We found that STAMBP reduces the binding affinity between RB1 and E2F1, whereas the association between RB1 and the E2F1 K161/164R mutant remained unaffected (Fig. 4 H, 4 I). Similarly, HDAC1, a core component of the RB1–E2F1 repressive complex, showed reduced binding to E2F1 upon STAMBP-mediated deubiquitination, confirming broader destabilization of the transcriptional inhibitory machinery (Fig. 4 J, 4 K). Given that K63-linked ubiquitin chains act as scaffolds stabilizing protein interactions, these findings support a model in which K63-linked polyubiquitination of E2F1 enhances its association with RB1, thereby locking E2F1 in a repressed state. Conversely, STAMBP-mediated deubiquitination disrupts this complex, activating target gene expression through a phosphorylation-independent mechanism. Pharmacological targeting of STAMBP suppresses proliferation and migration in bladder cancer cells Our prior research suggests that E2F1 activity plays a critical role in driving cellular proliferation( 14 ). Having established that STAMBP dismantles the RB1-E2F1 repressive complex via K63-linked deubiquitination of E2F1, we next assessed the functional consequence of pharmacologically inhibiting STAMBP activity in bladder cancer cells. To this end, we employed the STAMBP antagonist BC1471, which blocks its deubiquitinating enzyme activity( 26 ). As shown in Fig. 5 A, BC1471 selectively suppressed colony formation in STAMBP-overexpressing (STAMBP) cells, whereas blank vector control (BV) cells were minimally affected. Quantitative analysis (Fig. 5 B) confirmed a significant reduction in colony count specifically in STAMBP overexpressing groups (red bars). Consistent with this, BC1471 induced significant proliferation arrest in STAMBP overexpressing cells (Fig. 5 C, 5 D), and reduced cell migration without impacting BV controls. Proliferation assays (Fig. 5 E, 5 F) further confirmed BC1471 induced ​​selective growth arrest in STAMBP overexpressing cells​​. Collectively, these results demonstrate that pharmacological inhibition of STAMBP with BC1471 restores K63-linked ubiquitination of E2F1, thereby re-establishing RB1-imposed repression of E2F1 activity and thereby suppresses the proliferation and migration of bladder cancer cells. Depletion of STAMBP Suppresses Bladder Cancer Tumorigenesis In Vivo. To explore the role of STAMBP in bladder cancer tumorigenesis in vivo , we developed a subcutaneous xenograft model incorporating STAMBP-knockout and wild-type T24 cells. Tumors derived from STAMBP-knockout cells exhibited significantly slower growth, with marked reductions in both tumor volume and weight compared with the WT group (Fig. 6 A-C), indicating that the absence of STAMBP hinders the tumorigenic potential of bladder cancer cells. Furthermore, immunohistochemical (IHC) analysis confirmed a marked decline in the number of Ki67-positive proliferating cells and levels of STAMBP protein in STAMBP- knockout tumors (Fig. 6 D), supporting the conclusion that STAMBP deletion diminishes tumor cell proliferation in vivo . Mechanistically, western blot analysis of tumor tissues showed substantial downregulation of key E2F1 downstream targets, including CDC2 and DHFR, in STAMBP-knockout tumors relative to controls (Fig. 6 E). Collectively, these findings highlight STAMBP as a critical promoter of bladder cancer tumorigenesis, whose loss impedes tumor growth by downregulating E2F1-mediated proliferative pathways. Stambp Deficiency Lessens BBN‑Induced Bladder Tumorigenesis in Mice To clarify the tumorigenic role of STAMBP in bladder cancer in vivo , CRISPR/Cas9 strategy was employed to generate Stambp bladder conditionally knockout mice (Stambp flox/flox )(Fig. 7 A). We generated bladder-specific STAMBP knockout mice (Stambp flox/flox ; Upk2-Cre) by crossing Stambp fl/fl mice with Upk2-Cre transgenic mice, which express Cre recombinase under the urothelial-specific uroplakin 2 (Upk2) promoter. Genomic PCR confirmed the successful recombination of the Stambp locus in homozygous mice (Fig. 7 B). No significant differences in body weight were observed between Stambp flox/flox Upk2-Cre mice and Stambp fl/fl mice, indicating that Stambp deletion did not induce systemic toxicity (Fig. 7 C). To assess the impact of STAMBP deficiency on bladder tumorigenesis, we subjected Stambp flox/flox ; Upk2-Cre and control mice to a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced carcinogenesis model (Fig. 7 D). Beginning at five weeks of age, mice received 0.05% BBN in drinking water for 21 weeks. At the 26-week endpoint, bladder tissues were harvested for analysis. Mice lacking Stambp exhibited a notable reduction in tumor burden induced by BBN when compared to the control group (Fig. 7 E). Bladder weights, serving as a surrogate marker of tumor progression, were significantly lower in the Stambp flox/flox ; Upk2-Cre mice (Fig. 7 F). Histopathological evaluation revealed that 50% of tumors in knockout mice were in the early stages (Stage I), while 83% of tumors in the control group had progressed to more advanced stages (Stage II/III) (Fig. 7 G). Notably, STAMBP-deficient mice displayed significantly improved survival compared with controls (Fig. 7 H). Additionally, immunohistochemical (IHC) analysis highlighted a reduction in pathological damage in bladders lacking Stambp, with features such as diminished stromal invasion and maintained urothelial structure (Fig. 7 I). Collectively, these findings demonstrate that the urothelial-specific deletion of STAMBP mitigates BBN-driven bladder tumorigenesis, delays tumor progression, and improves survival. underscoring STAMBP as a critical mediator of bladder cancer pathogenesis in vivo . Based on our mechanistic insights, we propose a 'Dual-Lock' model that delineates two distinct pathways regulating RB1-E2F1 complex stability and E2F1 activation (Fig. 8 ): (i) the canonical phosphorylation-dependent pathway, where CDK4/6-mediated RB1 phosphorylation induces conformational change-mediated release of E2F1 from the repressive complex (RB1-E2F1-HDAC1), activating cell cycle targets (e.g., CCNE1, DHFR); and (ii) a deubiquitination-driven pathway, wherein K63-linked ubiquitin chains on E2F1 (K161/164) act as molecular scaffolds that reinforce complex stability. STAMBP—identified as the key K63-specific deubiquitinase—removes these chains, dismantling the scaffold and destabilizing the complex without affecting RB1 phosphorylation; this liberates transcriptionally active E2F1 to drive malignant progression. Together, the model provides a mechanistic framework for understanding context-specific E2F1 activation in bladder cancer pathogenesis. Discussion Bladder cancer represents a major global health burden. In 2025, it is projected that 17,420 deaths in the United States will be attributable to bladder cancer, representing the highest mortality among urological malignancies in men( 1 ). Bladder cancer develops through two distinct pathways, resulting in non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Standard therapies for NMIBC involves transurethral resection of the bladder tumor (TURBT) followed by intravesical Bacillus Calmette-Guérin (BCG), however, BCG failure occurs in approximately 40% of patients with high-risk disease​( 27 , 28 ). For MIBC, cisplatin-based neoadjuvant chemotherapy (NAC) remains the standard of care, yet up to 50% of patients with renal or cardiac comorbidities are ineligible( 29 )​​. These limitations underscore the urgent need for precision-based therapeutic approaches that can enhance treatment efficacy. Encouragingly, recent advances in molecular subtyping of bladder cancer have facilitated the development of targeted therapies, such as FGFR3 inhibitors, as transformative treatments for this disease( 3 , 30 , 31 ). Beyond FGFR3 inhibition, multidimensional targeting strategies encompassing Nectin-4-directed agents, immune checkpoint blockade (PD-1/PD-L1), HER2 pathway modulation, tumors with DNA repair deficiencies such as those harboring ERCC2 mutations, and novel epigenetic targets (e.g., BLM lactylation) have emerged as pivotal therapeutic avenues( 32 – 36 ). Nevertheless, these strategies do not address all patient populations, highlighting the continued need to identify additional therapeutic targets. As the important components of post translational modification, ubiquitin and de-ubiquitin family enzymes are involved in the initiation and progression of various diseases, including multiple cancers( 37 ). Modulating DUB activity has emerged as a promising therapeutic strategy to suppress tumor proliferation and improve responses to chemotherapy and immunotherapy( 38 ). Recent studies indicate several deubiquitinases, including BRCC3, OTUB1, and UCHL3, are aberrantly expressed in bladder cancer and play functional roles in its development( 39 – 43 ). In the present research, we identified STAMBP as another critical DUB gene associated with bladder cancer proliferation. By validating databases and tissue specimens, we demonstrate that STAMBP is highly expressed in bladder cancer. We conducted functional experiments and demonstrated that increasing levels of STAMBP facilitated the growth and invasion of BC cells, whereas the knockout of STAMBP hindered the aggressive phenotype of BC cells. Consistent with its oncogenic role, pharmacological inhibition of STAMBP by BC1471 (10 µM) significantly suppressed proliferation and migration in the STAMBP-high BC cells compared with control cells. Earlier research indicates that STAMBP enhances the migration and invasion of lung cancer cells and triple-negative breast cancer (TNBC) cells( 24 , 25 ). Consistent with these investigations, our results suggest that STAMBP represents a potential therapeutic target in bladder cancer. RNA sequencing combined with Gene Ontology (GO) enrichment analysis revealed a strong association between STAMBP and cell cycle regulation. Further analysis reveals a strong association between STAMBP and the E2F signaling pathway; this result has been validated through dual-luciferase reporter gene experiments. Members of the E2F signaling pathway act as transcription factors forming the central transcription axis( 44 ). Real-time quantitative PCR showed that STAMBP depletion significantly downregulates CDCA2, DHFR, and PCNA expression. The results from protein-level assays indicate that the loss of STAMBP leads to a decrease in the protein levels of CDCA2 and DHFR, while graded expression of STAMBP results in a dose-dependent increase in their protein levels. Together, these findings suggest that STAMBP positively regulates the transcriptional activator E2F1 within the E2F family. previous studies reported that elevated E2F1 expression in bladder cancer promotes progression from superficial to invasive stages( 10 ). Therefore, we primarily focus on the molecular mechanisms of E2F1 in our studies. The RB1–E2F1 axis represents the canonical model of E2F1 regulation. In this model, phosphorylation induces conformational changes in RB1 under specific conditions, releasing E2F1 to activate transcription of downstream genes( 45 ). Consequently, numerous studies have focused on the changes in the phosphorylation state of RB1. Additionally, many studies have concentrated on the post-translational modifications of E2F1, including phosphorylation, methylation, and acetylation( 46 – 48 ). Ubiquitination and deubiquitination, which are critical for protein homeostasis, also play important roles in E2F1 regulation, particularly through K63-linked ubiquitination. UCH37 is the first reported deubiquitinating enzyme capable of removing K63-linked ubiquitination from E2F1. Although this does not alter E2F1 protein stability, it enhances transcriptional activity through deubiquitination( 22 ). POH1 stabilizes E2F1 by removing both K63- and K11-linked ubiquitin chains, thereby preventing proteasomal degradation( 16 ). STAMBP has also been shown to directly bind to E2F1 and stabilize its expression in pancreatic cancer cells, possibly by inhibiting K48-linked polyubiquitination; however, this mechanism requires further experimental validation( 49 ). Despite these insights, it remains unclear whether ubiquitination enhances E2F1 stability, how this is related to RB1 phosphorylation, and whether it affects RB1-E2F1 binding. Our research demonstrates that STAMBP enhances the stability of E2F1. Laurence Dubrez et al., reported that lysine residues 161 and 164 of E2F1 are critical for its K63 polyubiquitination modification( 21 ). Therefore, we evaluated the effect of STAMBP on K63 ubiquitination of the E2F1 K161/164R mutant. The results showed that STAMBP treatment did not alter the K63 ubiquitination level of the E2F1 K161/164 mutant, indicating that STAMBP removes K63-linked ubiquitin chains to deubiquitinate E2F1. We also found that the addition of STAMBP weakens the interactions between RB1 and E2F1. We also examined the phosphorylation levels of RB1. We found that STAMBP does not affect RB1 phosphorylation. This observation raises an important question: if STAMBP does not influence RB1 phosphorylation, why is the interaction between E2F1 and RB1 reduced? Given that K63-linked ubiquitin chains act as scaffolds for protein-protein interactions, we propose a model where RB1-E2F1 binding is regulated by both RB1 pocket conformation and K63 polyubiquitination. K63 polyubiquitination reinforces the RB1-E2F1 interaction, whereas STAMBP-mediated removal of these chains disrupts binding, releasing E2F1. This release enhances downstream survival signaling, promoting the occurrence of bladder cancer. To further explore the potential of STAMBP as a therapeutic target, we conducted in vivo experiments. We first verified that the deletion of STAMBP inhibited the tumor-forming ability of bladder cancer cells in vivo using a nude mouse tumor model. In addition, we generated bladder-specific STAMBP knockout mice and employed a BBN-induced bladder carcinogenesis model. The results showed that the deletion of STAMBP increased the survival time of mice in the BBN-induced tumor model, while also reducing tumor volume and decreasing tumor progression severity. Transcription factors like E2F1 are appealing therapeutic targets. However, their broad functional pleiotropy and nuclear localization make tumor-specific inhibition particularly challenging. DUBs have recently emerged as promising druggable targets in oncology, with several inhibitors (e.g., PR-619 against USP14) currently undergoing clinical evaluation( 38 , 50 ). Moreover, Our study establish STAMBP as a pivotal oncogenic DUB in bladder cancer. We demonstrate that STAMBP destabilizes the RB1-E2F1 transcriptional repressor complex via K63-linked deubiquitination, thereby promoting cell-cycle progression and metastatic phenotypes. These findings provide a strong therapeutic rationale for targeting STAMBP. However, limitations remain. Current tissue analyses lack subtype-specific stratification and validation across multicenter cohorts. Additionally, translational challenges include optimizing BC1471's therapeutic window and pharmacodynamics in patient-derived xenograft models before clinical assessment. Future studies should also correlate STAMBP expression with chemotherapy response in Phase II trials, which may accelerate its development as a predictive biomarker. Conclusions Our study uncovers a phosphorylation-independent regulatory axis governing the RB1-E2F1 complex, mediated by the deubiquitinase STAMBP. We demonstrate that RB1-E2F1 complex integrity is co-regulated by classical phosphorylation mediates RB1 "pocket" conformation and K63-ubiquitin "Scaffold" of E2F1. Critically, we identify STAMBP as an oncogenic trigger and a therapeutic target for a molecularly defined subset of BC patients. Abbreviations AMSH Associated molecule with the SH3 domain of STAM BBN N-butyl-N-(4-hydroxybutyl)-nitrosamine BC Bladder cancer BCG Bacillus Calmette–Guérin CDK Cyclin-dependent kinase CDC2 Cell division control protein 2 DAB 3,3′-Diaminobenzidine DUB Deubiquitinating enzyme E2F1 E2F transcription factor 1 EGFR Epidermal growth factor receptor EMT Epithelial–mesenchymal transition FGFR Fibroblast growth factor receptor HAT Histone acetyltransferase HDAC Histone deacetylase JAMM JAB1/MPN/Mov34 metalloenzyme MIBC Muscle-invasive bladder cancer NAC Neoadjuvant chemotherapy NMIBC Non-muscle-invasive bladder cancer PKA Protein kinase A RB1 Retinoblastoma 1 STAMBP STAM-binding protein TMA Tissue microarray TURBT Transurethral resection of bladder tumor Declarations Data availability The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments We thank Professor Tian-Zhi Huang from Xiamen university and Will Fong from University of Kentucky for their numerous suggestions throughout the paper writing process. The schematic diagram (Fig.6J) was created using Figdraw. Funding This research was funded by grants from the National Natural Science Foundation of China (32270760), the Fundamental Research Funds for the National Key R&D Project (2022YFF0710700). Author information Authors’ Contributions T. Liu: Project administration, investigation, data curation, validation, visualization. QP. Shu: Investigation, supervision, validation, visualization. L. Yu: Investigation, validation, writing–original draft. CM. Jiang: Investigation, formal analysis, visualization. HH Tao: Investigation, formal analysis, software, Methodology. WZ Wang: Investigation, visualization. J Zhang: Investigation, data curation. YF Pang: Data curation, software. C. M: Formal analysis, data curation, supervision. HH. 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Regulation of E2F1 activity via PKA-mediated phosphorylations. Turkish journal of biology = Turk biyoloji dergisi 2020 ;44:215-29 Zhang W, Xu Z, Du Y, Liu T, Xiong Z, Hu J , et al. Identification of STAM-binding protein as a target for the treatment of gemcitabine resistance pancreatic cancer in a nutrient-poor microenvironment. Cell Death Dis 2024 ;15:657 Wu J, Liu C, Wang T, Liu H, Wei B. Deubiquitinase inhibitor PR-619 potentiates colon cancer immunotherapy by inducing ferroptosis. Immunology 2023 ;170:439-51 Additional Declarations There is a duality of interest Supplementary Files TableS1.pdf Table S1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":351673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmplification of STAMBP was associated with worse prognosis. \u003c/strong\u003eA, B. Expression of STAMBP in pan-cancer in the GEPIA and UALCAN database. C, D. Bladder cancer tissues versus normal tissues from Sanchez-Carbayo and Dyrskjot group in Oncomine databases. E. Analysis of overall survival for 402 individuals diagnosed with bladder cancer based on STAMBP expression in the GEPIA web server. The high STAMBP group showed significantly lower survival rates (\u003cem\u003eP\u003c/em\u003e = 0.027). F. Immunohistochemical staining of STAMBP in tissue microarrays, including urothelial cancer tissues and adjacent tissues. G. Analysis of tissue microarray data: STAMBP was expressed in 56 bladder cancer tissue samples and 10 corresponding adjacent tissue samples (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001). Statistical significance was analyzed by ANOVA or Student t test. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **P\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e ***P \u003c/em\u003e\u0026lt; 0.001, \u003cem\u003e****P \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/a5b4603c238ad3341aaf488f.png"},{"id":98624432,"identity":"ba9b7e3d-19ce-4859-82f9-b986e55d0ede","added_by":"auto","created_at":"2025-12-19 17:08:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":730019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression and knockout of STAMBP affect the proliferation, migration, and invasion of bladder cancer cells.\u003c/strong\u003e A. T24 cells were transfected with Flag-STAMBP or Flag-STAMBP\u003csup\u003eD348A\u003c/sup\u003e, and protein levels were detected by western blot. GAPDH served as a control. B. STAMBP protein levels in control and STAMBP-knockout T24 cells were detected by western blotting with GAPDH as a loading control. C. T24 cells were transfected with STAMBP or Flag- STAMBP\u003csup\u003eD348A\u003c/sup\u003e as indicated, and wild-type T24 cells served as control. Colony formation assays were performed for 11 days to detect cell viability. The colonies were stained with crystal violet and photographed. The number of colonies was counted and plotted (n = 3). D. Colony formation assays were performed for 8 days to show the viability of STAMBP-knockout T24 bladder cancer cells. Colonies were stained with crystal violet and subsequently imaged (left). The number of colonies was counted and plotted (right, n = 3). E. T24 cells were transfected with Flag- STAMBP or Flag-STAMBP\u003csup\u003eD348A\u003c/sup\u003e as indicated, and wild-type T24 cells served as control. And CCK-8 assays were used to analyze cell proliferation (n = 6). F. The cell proliferation ability of STAMBP-knockout cells was determined by CCK-8 assay (n = 6). G. T24 cells were transfected with Flag-STAMBP or FLAG-STAMBP\u003csup\u003eD348A\u003c/sup\u003e as indicated, and wild-type T24 cells served as control. Transwell experiments were used to evaluate the effects of STAMBP overexpression on cell migration. After seeding for 36 hours, the cells were imaged(left) and counted, and the results were plotted (right, n = 3). Scale bars, 100 μm. H. Transwell experiments were used to evaluate the effects of STAMBP deficiency on T24 bladder cancer cell migration. After seeding for 72 hours, the cells were imaged (left), images were captured and counted, and the results were plotted (right, n = 3). Scale bars, 100 μm. Data (mean ± SEM) are representative of 3 independent experiments. Statistical significance was analyzed by ANOVA or Student’s t-test. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **P\u003c/em\u003e \u0026lt; 0.01,\u003cem\u003e ***P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/bbe55147406c32287b9591d2.png"},{"id":98625485,"identity":"c8d202e9-8788-4a11-a816-de3750370f4b","added_by":"auto","created_at":"2025-12-19 17:09:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2255960,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAMBP activates the E2F signaling pathway.\u003c/strong\u003e A. KEGG enrichment bubble maps show that multiple signaling pathways are enriched in wild-type cells compared to STAMBP\u003csup\u003e-/- \u003c/sup\u003ecells. B. GSEA identified cell cycle and E2F pathway-related gene sets enriched in the wild-type cells. C. GSEA identified cell cycle and E2F pathway-related gene sets enriched in bladder cancer patients with high STAMBP expression. D. Luciferase pathway screening revealed that STAMBP significantly promoted E2F pathway activation in HEK293T cells (n = 3). E. STAMBP increases transcriptional activity of E2F signaling. E2F reporter firefly luciferase plasmid (300 ng), CMV (5 ng), and the indicated amounts of STAMBP plasmid were transfected into HEK293T cells. Reporter assays were performed 48 hours after transfection, and the results are presented as E2F/CMV luciferase activity. F. The positive correlation between STAMBP and cell cycle and E2F-targeted genes (CDCA2, DHFR, and PCNA) from the cBioPortal online tool. G. STAMBP loss inhibited the transcription of cell cycle and E2F‑targeted genes in T24 cells. The expression levels of the indicated cell cycle and E2F‑targeted genes were examined by real-time PCR in STAMBP knockout T24 cells and wild‑type T24 cells. H. STAMBP overexpression enhanced the transcription of cell cycle and E2F-targeting genes in T24 cells. The expression levels of the indicated cell cycle and E2F‑targeted genes were examined by real-time PCR in STAMBP\u003csup\u003e+/+\u003c/sup\u003e T24 cells and wild‑type T24 cells. I. The protein levels of the indicated cell cycle and E2F-targeted genes in STAMBP T24 cells were determined by western blot. J. The protein levels of the indicated cell cycle and E2F-targeted genes in STAMBP-overexpression cells were determined by western blot. The data are presented as the means ± SEM. Statistical significance was analyzed by ANOVA. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **P \u003c/em\u003e\u0026lt; 0.01,\u003cem\u003e ***P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/eca76cb8b4ec1da9665fcc98.png"},{"id":98625113,"identity":"33702b09-34e4-4e26-b4db-8f887ef5fe75","added_by":"auto","created_at":"2025-12-19 17:08:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":505208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTAMBP dismantles the RB1-E2F1 complex via K63 deubiquitination of E2F1. \u003c/strong\u003eA, B, C. The interaction between STAMBP and E2F1/HDAC1/RB1 were examined by immunoprecipitation in HEK293T. D. The interaction of STAMBP and E2F1 were examined by immunoprecipitation in T24 cells. E. The ubiquitination of E2F1 was assessed in the presence of STAMBP or the STAMBP\u003csup\u003eD348A\u003c/sup\u003e mutant. F. STAMBP ubiquitination level in the presence of E2F1 or the E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutant. G. Levels of ubiquitination of STAMBP at the K63-specific ubiquitin chain site in the presence of E2F1 or E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutants. H, I. The interaction of E2F1 and RB1 in the presence of STAMBP or STAMBP\u003csup\u003eD348A\u003c/sup\u003e mutants. J, K. The interaction of STAMBP and HDAC1 in the presence of E2F1 or E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutants. I. The binding level of STAMBP to RB1 protein in the presence of E2F1 or E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutants.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/7cc1adf77b6c89dc6a7478bc.png"},{"id":98526776,"identity":"d88d08be-6835-4f95-bc36-ebfd8fc059a1","added_by":"auto","created_at":"2025-12-18 14:44:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":442649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBC1471 mitigates STAMBP upregulation.\u003c/strong\u003eA, B. The clone formation assay and the statistical chart represent that STAMBP inhibitor BC1471 attenuates clone formation proliferation in a concentration-dependent manner (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01). 11 days after the cells were seeded, the colonies were stained with crystal violet and photographed. C, D. Transwell assay and the statistical chart represent that STAMBP inhibitor BC1471 attenuates migration viability in a concentration-dependent manner (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01). 36 hours after the cells were seeded, the cells were stained with crystal violet and photographed. Scale bars, 100 μm. E, F. The CCK-8 assays represent that STAMBP inhibitor BC1471 attenuates T24 cell growth. Data are presented as the mean ± SEM. Statistical significance was analyzed by ANOVA or Student’s \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **P \u003c/em\u003e\u0026lt; 0.01,\u003cem\u003e ***P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/d5d1d1d26763d926c6a63f7c.png"},{"id":98526778,"identity":"93dc872b-cfcc-48e1-b69e-0bb2965332ab","added_by":"auto","created_at":"2025-12-18 14:44:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":722357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of STAMBP suppresses bladder cancer tumorigenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Xenograft models were established by subcutaneously injecting wild-type or STAMBP knockout\u003csup\u003e \u003c/sup\u003ecells and last for 5 weeks (n = 5). The mice were executed, and tumors were separated and weighed. A. Tumors were extracted from euthanized mice and representative images were shown. B. Quantitative results of tumor weight. C. Tumors were measured 3 times per week, and the tumor volume was plotted. D. H\u0026amp;E, STAMBP, Ki67 staining of tumor tissue from the xenograft model. Scale bars, 100 μm. Statistical analysis of the tumor volume and weight were performed using one-way ANOVA. E. Detection of related protein indices in tumor tissue samples via western blotting. The means ± standard error of three independent experiments is shown. \u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05\u003cem\u003e, **P \u003c/em\u003e\u0026lt; 0.01\u003cem\u003e, ***P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/60e4906c757d05fabe404bed.png"},{"id":98625526,"identity":"a2a77077-3fe1-47ee-95b6-4dd34ae20be7","added_by":"auto","created_at":"2025-12-19 17:09:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":684392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStambp deficiency attenuates BBN-induced bladder tumorigenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eA. CRISPR/Cas9-mediated knockout strategy for generating bladder-specific Stambp conditional knockout mice (Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre). B. Genomic PCR validation of Stambp recombination in bladder tissues from Stambp\u003csup\u003eflox/flox\u003c/sup\u003e and Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre mice. C. Body weight trajectories of Stambp\u003csup\u003eflox/flox\u003c/sup\u003e and Stambp\u003csup\u003eflox/flox;\u003c/sup\u003e Upk2-Cre mice during BBN treatment D. Experimental timeline of BBN carcinogenesis model. Tamoxifen-induced recombination (3 weeks) precedes BBN exposure (5-26 weeks). E. Histopathological grading reveals delayed tumor progression in Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre mice (50% Stage I) versus controls (83% Stage II/III). F. Bladder weight examination data of bladder tissues. G. Histopathological grading. H. Overall survival of Stambp\u003csup\u003eflox/flox\u003c/sup\u003e (n = 8) and Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre (n = 7) mice in the BBN‑induced bladder cancer model. I. Mice bladder IHC of E2f1, Cdc2 and Dhfr from Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre mice and the control group. Scale bars, 100 μm.\u003cem\u003e*P \u003c/em\u003e\u0026lt; 0.05,\u003cem\u003e **P \u0026lt; \u003c/em\u003e0.01,\u003cem\u003e ***P \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/cc15c656bd153749ba67653f.png"},{"id":98526782,"identity":"1edbd42a-4cf6-40e5-8c2f-d698d223418f","added_by":"auto","created_at":"2025-12-18 14:44:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":167644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\"Dual-Lock\" model for RB-E2F1 complex regulation.​​ \u003c/strong\u003e(Top)​​ ​​Canonical phosphorylation-dependent activation​​: CDK4/6-mediated RB phosphorylation induces allosteric changes, triggering E2F1 release from the repressive complex (RB-E2F1-HDAC1) and activating downstream targets (e.g., CDK2, PCNA, DHFR). (Bottom)​​ ​​Non-canonical K63 deubiquitination-dependent activation​​: K63-linked ubiquitin chains (K63Ub) on E2F1 function as ​​molecular scaffolds​​ reinforcing complex stability. STAMBP removes these chains, dismantling the scaffold and destabilizing the complex ​​without involving RB1 phosphorylation modifications​​, thereby liberating transcriptionally active E2F1. Together, these parallel pathways exemplify the dual regulatory locks controlling E2F1 activity.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/ca11c562a612bae2b5583840.png"},{"id":100379930,"identity":"607ef1d2-02a1-40b4-bec7-6fc76c991f3c","added_by":"auto","created_at":"2026-01-16 09:55:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7026487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/8f0c5b9e-2564-49d5-b669-7dc43d763bbf.pdf"},{"id":98526770,"identity":"2a7c31b4-2b93-4551-97b7-629e2cfa1057","added_by":"auto","created_at":"2025-12-18 14:44:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":44590,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8327775/v1/57768e784bbea773c1b79235.pdf"}],"financialInterests":"There is a duality of interest","formattedTitle":"STAMBP-Mediated K63 Deubiquitination of E2F1 Release E2F1 from RB Repressive Complex to Drive Bladder Cancer Progression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer (BC) is the most common malignant tumor of the urinary system and is projected to cause 17,420 deaths among 84,870 new cases in the United States in 2025(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Globally, BC exhibits a pronounced gender disparity, with incidence rates in men being two- to four-fold higher than in women(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The therapeutic strategy has rapidly evolved from surgery-chemotherapy combinations to precision approaches that incorporate molecularly targeted agents(e.g., \u003cem\u003eErdafitinib\u003c/em\u003e for FGFR3/2-altered tumors), immune checkpoint inhibitors (e.g., \u003cem\u003epembrolizumab\u003c/em\u003e), and antibody-drug conjugates(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Notably, pembrolizumab significantly improved median overall survival to 16 months compared with 9 months for chemotherapy in cisplatin-ineligible patients(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), while Erdafitinib extended survival to 12.1 months versus 7.8 months in chemotherapy-treated controls (HR\u0026thinsp;=\u0026thinsp;0.64; 95% CI: 0.47\u0026ndash;0.88)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Critically, actionable targets such as FGFR alterations are present in only 15\u0026ndash;20% of patients(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), leaving\u0026thinsp;\u0026gt;\u0026thinsp;80% without molecularly guided therapies. Consequently, further research into the molecular mechanisms of BC and the identification of broader therapeutic targets are urgently needed to improve patient outcomes.\u003c/p\u003e \u003cp\u003eE2F transcription factor 1 (E2F1), a master regulator of cell cycle-dependent transcription, governs diverse cellular processes including apoptosis, DNA damage repair, and metabolism(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Dysregulation of E2F1 has been observed in multiple cancer types(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Notably, E2F1 expression is particularly critical for progression from superficial to invasive disease. Molecular subtyping reveals that T1-luminal genomically unstable tumors exhibit significant enrichment of E2F1 transcription factor motifs (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Overexpression of E2F1 drives the transition from superficial to invasive stages by activating cell cycle targets, such as CDK1 and CCNE1, through promoter motif enrichment(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In addition, E2F1 activity is critically regulated by direct binding of the retinoblastoma protein (RB1). During G1 phase, hypo-phosphorylated RB1 sequesters E2F1 in a repressive complex, inhibiting transcriptional activation of S-phase genes until CDK-mediated RB1 phosphorylation result in a conformational change to release E2F1. This release allows E2F1 to function as an active transcription factor that promotes the expression of genes essential for entering the S phase(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). E2F1\u0026rsquo;s transcriptional activity can be modulated by its association with co-activators such as histone acetyltransferases (HATs) and co-repressors such as histone deacetylases (HDACs)(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Beyond this canonical model, tumors frequently exhibit continuous E2F1 activation independent of RB1 phosphorylation, suggesting an alternative RB1 conformation\u0026ndash;independent regulatory mechanism. The emerging research highlights the critical role of post-translational modifications (PTMs) in modulating E2F1 stability and activity.​ For instance, ubiquitination pathways significantly regulate E2F1 proteostasis: APC/C Cdh1 mediates K11-linked degradation, whereas POH1 stabilizes E2F1, promoting survival signals like Survivin and FOXM1 during tumorigenesis(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, current studies focus predominantly on PTM-mediated effects on E2F1 stability or transcriptional activity, without addressing potential crosstalk with the RB1-E2F1 complex dynamics.​​ Specifically, whether and how specific PTMs directly disrupt or reinforce the RB1-E2F1 interaction remains unexplored, highlighting a critical gap in understanding PTM-driven regulation of this core cell cycle machinery.\u003c/p\u003e \u003cp\u003eProtein homeostasis is dynamically regulated by PTMs, especially ubiquitination, which involves E1\u0026ndash;E2\u0026ndash;E3 ligase\u0026ndash;mediated substrate tagging and removal of ubiquitin chains by deubiquitinating enzymes (DUBs). Dysregulation of these processes significantly contributes to oncogenesis(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The ubiquitin molecule can form connections through seven distinct lysine sites (K6, K11, K27, K29, K33, K48, and K63)(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Each conferring distinct functional consequences. K63-linked ubiquitination can influence various protein characteristics, including protein interactions, translocation, and activation processes(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). For example, TRAF2 K63-linked ubiquitination mediates interactions with TAB2/3 and activates the downstream kinases IKK and JNK(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). K63-linked ubiquitination of E2F1 has also been reported. The E3 ubiquitin ligase cIAP1 catalyzes K63-linked ubiquitination of E2F1 at lysines 161 and 164 (K161/K164). These specific residues are essential for E2F1-mediated gene activation(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Lin et al. identified UCH37 as the first DUB that directly regulates E2F1 activity. They proposed the following model: E2F1 is ubiquitinated with K63-linked ubiquitin chains, and this modification represses its transcriptional activity. UCH37 alleviates this repression by removing K63-linked ubiquitin chains from E2F1(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The molecular basis of why K63-linked ubiquitination suppresses E2F1 activity, however, remains unresolved.\u003c/p\u003e \u003cp\u003eThe STAM-binding protein (STAMBP), also known as AMSH (associated molecule with the SH3 domain of STAM), is a zinc-dependent metalloprotease that belongs to the JAMM/MPN family of DUBs. This enzyme specifically hydrolyzes K63-linked polyubiquitin chains, thereby modulating endosomal sorting and signal transduction pathways(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Recent studies have shown that STAMBP is closely related to the occurrence and development of many diseases, and STAMBP has an important role in the field of oncology. In triple-negative breast cancer, high levels of STAMBP were correlated with poor prognosis. STAMBP stabilized the RAI14 protein by suppressing the K48-linked ubiquitination of RAI14, thereby preventing its proteasomal degradation(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In lung adenocarcinoma, it regulates EGFR ubiquitination to activate pro-EMT signaling(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Despite these findings, the pathological role of STAMBP in bladder cancer remains entirely unknown.\u003c/p\u003e \u003cp\u003eIn the present study, we integrated multi-platform analyses and identified significant upregulation of STAMBP in bladder tumors. Elevated STAMBP expression was associated with reduced disease-free survival, supporting its prognostic relevance. The pathological overexpression of STAMBP in malignant epithelia was confirmed by immunohistochemical validation in clinical cohorts. Functionally, we demonstrate that STAMBP interacts with E2F1 and selectively suppresses K63-linked polyubiquitination to enhance E2F1 transcriptional activity. Crucially, K63-linked ubiquitin chains serve as molecular scaffolds that stabilize the RB1-E2F1 complex, and their deubiquitination triggers dissociation of the repressor-transcription factor complex, liberating transcriptionally active E2F1. Based on these findings, we propose a 'Dual-Lock' mode. In this model, RB1-E2F1 binding affinity is co-regulated by two mechanisms: the canonical RB1 pocket conformation acting as a gatekeeper, and K63-polyUb chains functioning as allosteric stabilizers at the protein-protein interface. This model addresses the long-standing question of constitutive E2F1 activation in cancers with intact RB1 and establishes K63-linked ubiquitination as a critical rheostat of cell cycle control.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture and Cell Lines\u003c/h2\u003e \u003cp\u003eCell lines including T24 and HEK293T cell lines were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (CASS). HEK293T cells were cultured in DMEM medium and T24 cells were cultured in McCoy's 5A medium. All culture medium were supplemented with 10% fetal bovine serum (FBS; Gibco, China), 100 U/ml penicillin-G sodium, and 100 mg/ml streptomycin sulfate. All cells were cultured in an incubator maintained at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents and Antibodies\u003c/h3\u003e\n\u003cp\u003eGAPDH (Cat. #60004-1-Ig) were purchased from Proteintech; FLAG (Cat. #M185-6), MYC (Cat. #M047-3), HA (Cat. #M180-3) were purchased from MBL (Medical \u0026amp; Biological Laboratories Co., Ltd.); Antibodies against STAMBP (Cat. #5245) and E2F1 (Cat. #3742) for immunoprecipitation (IP) were obtained from Cell Signaling Technology; Antibodies against STAMBP (Cat. #A7065), E2F1 (Cat. #A16720), CDC2 (Cat. #28439) for immunohistochemistry (IHC) were purchased from Abclonal (Abclonal, United States); Antibodies against DHFR (Cat. #43497) for immunohistochemistry (IHC) were purchased from Cell Signaling Technology; BC1471(HY-122883) were purchased from Medchemexpress. Tissue microarrays was purchased from Shanghai Outdo Biotech Company (Cat. HBlaU066Su01).\u003c/p\u003e\n\u003ch3\u003eGenetic Knock-Out of STAMBP in T24 Cells\u003c/h3\u003e\n\u003cp\u003eThe STAMBP gene knockout in T24 cells was conducted utilizing the CRISPR-Cas9 technology. In summary, we designed sgRNAs for STAMBP using the online tool available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/\u003c/span\u003e\u003cspan address=\"http://crispor.tefor.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Subsequently, the sgRNA was ligated into the lentiCRISPRv2 vector (Addgene, USA). The resulting vector containing the gRNA was co-transfected with packaging plasmids into HEK293T cells to facilitate virus packaging. The supernatant, which contained viral particles, was collected and utilized to infect T24 cells. Following 48 hours of infection, positive cells were selected through puromycin (1 \u0026micro;g/ml) treatment. Approximately 5 days later, the positive clones were distributed into 96-well plates using a gradient dilution method. After 14 days, single clones were picked. A portion of the screened monoclonal cells was utilized for protein extraction to conduct western blotting experiments aimed at verifying the successful knockout of STMBP. The sgRNA sequences: sgRNA-F: 5\u0026prime;-caccGGATAATCTCAACTCCAGAG-3\u0026prime;, sgRNA-R e: 5\u0026prime;-aaacCTCTGGAGTTGAGATTATCC-3\u0026prime;.\u003c/p\u003e\n\u003ch3\u003ePlasmids and sgRNA\u003c/h3\u003e\n\u003cp\u003eFlag-STAMBP, Flag-STAMBP\u003csup\u003eD348A\u003c/sup\u003e HA-HDAC1, Myc-RB1, HA-E2F1, Myc-E2F1, HA-E2F1\u003csup\u003eK161/164R\u003c/sup\u003e. To overexpress STAMBP, we purchased the overexpression plasmids for STAMBP from Miao Ling Biotech\u0026rsquo;s plasmid platform. The sgRNA sequences: sgRNA-F, 5'-caccGGATAATCTCAACTCCAGAG-3'; sgRNA-R, 5'-aaacCTCTGGAGTTGAGATTATCC-3'.\u003c/p\u003e\n\u003ch3\u003eCCK8 assays\u003c/h3\u003e\n\u003cp\u003eCells were seeded in a 96-well plate at a density of 2000 cells per well. Testing was conducted every 24 hours over a period of 4 days. At each time point, 10 \u0026micro;l of CCK8 (Biosharp, Wuhan, China) was mixed with 100 \u0026micro;l of culture medium and added to the test wells. The plate was subsequently incubated at 37\u0026deg;C for 2 hours, and the absorbance was measured at OD450 nm using a microplate reader.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assays\u003c/h2\u003e \u003cp\u003eCells were seeded in a 6-well plate at a density of 1200 cells per well. The plates were incubated at 37\u0026deg;C for 10 days. Then the colonies were fixed using 4% paraformaldehyde for 30 minutes and stained with a 0.2% crystal violet solution for an additional 30 minutes. The clones were photographed and counted.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranswell Migration Assays\u003c/h3\u003e\n\u003cp\u003e500 \u0026micro;l culture medium containing 20% FBS was added to the lower chamber. Serum-free medium was placed in the upper chamber. Cells were seeded in the upper chamber. After 24 hours of incubation, the migrated cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Photographs were taken, and the cells were counted.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and real-time PCR\u003c/h3\u003e\n\u003cp\u003eTrizol reagent (Ambion, USA) was used to isolate total RNA. The ABScript II RT Master Mix (Tesingke, Wuhan, China) was used to synthesized cDNA following the manufacturer\u0026rsquo;s protocol. The real-time PCR analysis was conducted on a Bio-Rad CFX96 instrument (Hercules, CA, USA) and the quantification was performed utilizing GraphPad Prism 8. The primer sequences used are listed in Table S1.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were collected and lysed using RIPA buffer containing Phosphorylase Inhibitors (Biosharp, Wuhan, China) and Protease Inhibitors (Biosharp, Wuhan, China).The total proteins form each group were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Shanghai, China). After treatment with 5% skim milk for 1 hour at room temperature, the PVDF membrane was incubated with the primary antibody overnight at 4\u0026deg;C. Following three washes with TBST, the membrane was incubated with secondary antibody for 2 hours at room temperature and washed with TBST. The protein expressions on the PVDF membrane were detected using enhanced chemiluminescence (Biosharp, Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue Microarray and Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe Tissue Microarray (Outdo Biotech, Shanghai, China) comprises of 116 bladder cancer tissues and 46 adjacent non-cancerous tissues. The slides were treated with a 10% solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and subsequently permeabilized using 0.1% TX-100. Following the reagent supplier's protocol. the primary and secondary antibodies were applied to the slides for incubation. The DAB protein coloring solution was then used to stain the protein expression, which was observed under a microscope. The Histoscore was calculated by multiplying the staining intensity score by the positivity rate observed in each tissue sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003e5 ng of the pRL-CMV plasmid, 300 ng of E2F-Luc vectors (Yeasen, China) and appropriate amount of Flag-STAMBP plasmid or an empty vector were transfected into T24 cells or HEK293T cells. After 48 hours, luciferase activity was measured using a dual-luciferase assay kit (Promega), with Promega luciferase system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCoimmunoprecipitation (Co‑IP) assay\u003c/h2\u003e \u003cp\u003eCells were lysed in ice-cold lysis buffer (30 mM Tris\u0026ndash;HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 10 \u0026micro;g/mL aprotinin, 10 \u0026micro;g/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). The resulting lysates were subjected to centrifugation at 12,000 rpm for 10 minutes at 4\u0026deg;C, the cell debris was discarded, and the cell supernatant was retained. G-agarose beads (Smart-Lifesciences, Changzhou, China) precoated with the indicated antibodies were added to the supernatant with lysed cells, which were incubated at 4\u0026deg;C. Six hours later, the Co-IP proteins were subjected to Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDeubiquitination analysis\u003c/h2\u003e \u003cp\u003eHEK293T cells were transfected with indicated plasmids, and 24 hours post-transfection, the cell culture medium was replaced with a fresh culture medium containing10 \u0026micro;M of MG132. At 36 hours post-transfection, the cells were washed using PBS and lysed with SDS lysis buffer (10% SDS in PBS). The lysates were heated to 95\u0026deg;C, followed by the addition of a twofold volume of modified RIPA buffer (50 mM Tris\u0026ndash;HCl pH 7.4, 150 mM NaCl, 1 mM EDTA and protease inhibitor). Subsequently, the lysates were cooled on ice for 30 min and centrifuged at 12,000\u0026times;g for 15 min at 4\u0026deg;C. Finally, the supernatant was subjected to anti-HA immunoprecipitation and Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003eThe prepared tissue sections were deparaffinized through consecutive treatments with xylene and varying concentrations of alcohol, with each immersion lasting 5 minutes. Mayer's hematoxylin (Wako, Japan) was utilized for nuclear counterstaining, which was subsequently followed by treatment with a 1% eosin solution for additional counterstaining. Before being observed under an Olympus microscope (Tokyo, Japan), the sections were cleared and dehydrated with the use of alcohol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnimal study\u003c/h2\u003e \u003cp\u003eTumor Xenografts: Four-week-old nude mice were purchased from the Experimental Animal Center of Wuhan University (Wuhan, China). Following a week of acclimatization, the mice were subcutaneously injected with 4 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells. Tumors were measured every other day after the appearance of subcutaneous tumors. The volume of the tumors was determined using the formula: volume = (length\u0026times;width\u003csup\u003e2\u003c/sup\u003e) \u0026times; 0.5. After 31 days of observation, the nude mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, and the transplanted tumors were taken out in a biosafety cabinet. Each group of tumors was imaged, and both their volume and weight were quantified. The tumor tissues were fixed with 4% paraformaldehyde. and subsequent staining was carried out using either HE or IHC techniques.\u003c/p\u003e \u003cp\u003eAOM/DSS-induced colorectal cancer model: BALB/c mice aged 5\u0026ndash;6 weeks were obtained from the Experimental Animal Center of Wuhan University (Wuhan, China). Upk2-Cre and Stambp\u003csup\u003eflox/flox\u003c/sup\u003e mice were procured from the Model Animal Research Center of Nanjing University. Upk2-Cre; Stambp\u003csup\u003eflox/flox\u003c/sup\u003e and Stambp\u003csup\u003eflox/flox\u003c/sup\u003e male mice were generated through selective breeding. The mice (5 weeks old) were administered tamoxifen at a dosage of 75 mg/kg for 5 days. Three weeks later, the mice were provided with drinking water with or without 0.05% BBN. Mouse weights were monitored every 3 days. Mice were sacrificed in the 26th week, and both tumor weights and volumes were recorded. Additionally, all mice underwent pathological examination. The Survival percentage rate of tamoxifen-treated mice was also evaluated.\u003c/p\u003e \u003cp\u003eAll mice were maintained under SPF conditions with a 12:12 h dark: light cycle. All efforts were taken to reduce the suffering of the animals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDatabase analysis\u003c/h2\u003e \u003cp\u003eThe TIMER, GEPIA, and UALCAN databases were employed to analyze the expression of STAMBP and assess its prognostic significance. The clinical relevance of STAMBP in bladder cancer patients was evaluated using the Sanchez-Carbayo Bladder 2 and Dyrskjot Bladder 3 datasets. These datasets were sourced from Oncomine (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.oncomine.org\u003c/span\u003e\u003cspan address=\"https://www.oncomine.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We applied the log-rank test and Cox regression analyses to generate Kaplan\u0026ndash;Meier curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical significance of the entire dataset was assessed using GraphPad Prism software. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM values were used to present all data. Student's \u003cem\u003et\u003c/em\u003e-tests or one-way ANOVA were conducted for statistical analysis, considering \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 as the threshold for determining statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAmplification of STAMBP was associated with worse prognosis\u003c/h2\u003e \u003cp\u003eTo examine the expression of STAMBP in bladder cancer, we analyzed datasets from GEPIA, UALCAN, and the Sanchez-Carbayo and Dyrskjot cohorts. The results revealed that STAMBP expression was significantly higher in bladder cancer tissues than in normal bladder tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Further analysis of the GEPIA database showed a negative correlation between high STAMBP expression and disease-free survival in patients with bladder cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting that elevated STAMBP may serve as an independent prognostic marker of poor outcomes. To validate these findings, immunohistochemical assessments was performed using tissue microarrays comprising 56 bladder cancer tissues and 10 normal bladder tissues. The results from the tissue microarray analysis indicated that STAMBP expression was significantly higher in bladder cancer tissues than in adjacent normal paracancerous tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Collectively, these results highlight the potential oncogenic role of STAMBP in the development and progression of bladder cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression and knockout of STAMBP affect the proliferation, migration, and invasiveness of bladder cancer cell lines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the function of STAMBP in bladder cancer cells, T24 cells were transfected with either a FLAG-STAMBP (wild-type) plasmid or a FLAG-STAMBP\u003csup\u003eD348A\u003c/sup\u003e-a catalytic-inactive mutant(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The expression levels of STAMBP were subsequently evaluated by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, T24 cell lines lacking STAMBP (KO1/KO2) were created using the CRISPR/Cas9 approach, and the absence of STAMBP expression was validated by western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To assess cell viability and tumorigenic potential, both colony formation and cell growth assays were performed. The results showed that the ectopic expression of STAMBP enhanced colony formation and increased viability of T24 cells in comparison to the parental T24 cells. Conversely, transfection with STAMBP\u003csup\u003eD348A\u003c/sup\u003e had no effect on either colony formation or the proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, E). Consistently, wild-type (WT) T24 cells displayed robust colony-forming capacity, whereas the STAMBP-knockout T24 cells generated considerably fewer colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, F). Furthermore, Transwell assays further revealed that STAMBP overexpression enhanced cell migration, whereas the mutation at the deubiquitination-catalyzing site in STAMBP did not alter migratory ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Conversely, STAMBP deficiency inhibited the migration of T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Collectively, these findings demonstrate that loss of STAMBP adversely affects the proliferation, viability, and migration of T24 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSTAMBP activates the E2F signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate the how STAMBP downregulation affects bladder cancer cell proliferation and migration, we performed RNA-Seq analysis using T24 and STAMBP-knockout T24 cells. KEGG pathway enrichment analysis was conducted to revealed that the cell cycle pathway was significantly enriched in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Gene Set Enrichment Analysis (GSEA) results indicated that the cell cycle and E2F signaling pathways were significantly enriched in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, we analyzed bladder cancer samples from the TCGA database, dividing patients into high and low expression groups based on STAMBP expression levels. Then, we conducted GSEA analysis, which corroborated the cellular findings, demonstrating significant enrichment of the cell cycle and E2F signaling pathways in bladder cancer tissues with high STAMBP expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To verify this result, we performed a luciferase signaling pathway assay in HEK293T cells to determine whether STAMBP could activate the relevant signaling pathways. The findings indicated that STAMBP can activate several signaling pathways, with the E2F1 pathway exhibiting the strongest and dose-dependent activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). We analyzed the relationship between STAMBP and cell cycle and E2F-targeted genes using the cBioPortal database, which revealed certain correlations between STAMBP and the genes CDCA2, DHFR, and PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). To validate these findings, we employed real-time quantitative PCR and Western blotting techniques. STAMBP knockout markedly reduced mRNA levels of cell cycle and E2F-targeted genes (CDC2, CCNA, CCND3, DHFR, E2F2, PCNA, and RNAGAP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Conversely, in T24 cells overexpressing STAMBP, there was a marked increase in the mRNA levels of these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Additionally, Western blotting results demonstrated that the absence of STAMBP resulted in decreased protein levels of CDC2 and DHFR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), and increased expression of both proteins under STAMBP overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Together, these results demonstrate that STAMBP positively regulates the cell cycle and E2F signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eSTAMBP dismantles the RB1-E2F1 complex via K63 deubiquitination of E2F1\u003c/h2\u003e \u003cp\u003eTo further explore the mechanism by which STAMBP activates the E2F pathway, we first tested the phosphorylation level of RB1, the result revealed that STAMBP does not alter RB1 phosphorylation status (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), indicating a phosphorylation-independent regulatory mechanism. Given that STAMBP is a K63 specific deubiquitinase and according to previous study, we hypothesis that STAMBP enhances the transcriptional activity by releasing it from RB1 through cleavage of the K63-linked ubiquitin chain. Firstly, the Co-IP experiments were performed to test the connection between E2F1 complex and STAMBP. The results demonstrated that STAMBP interacted with E2F1, RB1, and HDAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Notably, E2F1 exhibited particularly strong binding with STAMBP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Endogenous Co-IP experiments in T24 cells further confirmed that STAMBP interacts with E2F1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Subsequently, we aimed to investigate whether the ubiquitination level of E2F1 is regulated by STAMBP. The results demonstrated that STAMBP significantly reduced the ubiquitination of E2F1, while the STAMBP\u003csup\u003eD348A\u003c/sup\u003e had no effect, indicating that E2F1 ubiquitination depends on STAMBP deubiquitinating activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To determine whether K63-linked polyubiquitination is specifically removed from E2F1 by STAMBP, we employed an E2F1K161/164R double mutant that blocks. This mutant blocks K63-linked polyubiquitination at known regulatory sites critical for E2F1 stability(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, STAMBP expression reduced K63-linked ubiquitination in wild-type E2F1, whereas its inhibitory effect was ​​significantly reduced​​ on the mutant. Additionally, this site-specific regulation was further validated using a K63-only ubiquitin mutant (K63O), which forms exclusively K63-linked chains. The results indicated that in the presence of STAMBP, the K63 ubiquitination level of E2F1 was significantly reduced; however, while ubiquitination of the E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutant remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). These results indicate that STAMBP decreases K63-linked ubiquitination of E2F1 by targeting lysine residues 161 and 164. To investigate whether STAMBP-mediated removal of K63-linked ubiquitin from E2F1 affects its interaction with RB1, we conducted Co-IP. We found that STAMBP reduces the binding affinity between RB1 and E2F1, whereas the association between RB1 and the E2F1\u003csup\u003eK161/164R\u003c/sup\u003e mutant remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Similarly, HDAC1, a core component of the RB1\u0026ndash;E2F1 repressive complex, showed reduced binding to E2F1 upon STAMBP-mediated deubiquitination, confirming broader destabilization of the transcriptional inhibitory machinery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Given that K63-linked ubiquitin chains act as scaffolds stabilizing protein interactions, these findings support a model in which K63-linked polyubiquitination of E2F1 enhances its association with RB1, thereby locking E2F1 in a repressed state. Conversely, STAMBP-mediated deubiquitination disrupts this complex, activating target gene expression through a phosphorylation-independent mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003ePharmacological targeting of STAMBP suppresses proliferation and migration in bladder cancer cells\u003c/h2\u003e \u003cp\u003eOur prior research suggests that E2F1 activity plays a critical role in driving cellular proliferation(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Having established that STAMBP dismantles the RB1-E2F1 repressive complex via K63-linked deubiquitination of E2F1, we next assessed the functional consequence of pharmacologically inhibiting STAMBP activity in bladder cancer cells. To this end, we employed the STAMBP antagonist BC1471, which blocks its deubiquitinating enzyme activity(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, BC1471 selectively suppressed colony formation in STAMBP-overexpressing (STAMBP) cells, whereas blank vector control (BV) cells were minimally affected. Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) confirmed a significant reduction in colony count specifically in STAMBP overexpressing groups (red bars). Consistent with this, BC1471 induced significant proliferation arrest in STAMBP overexpressing cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), and reduced cell migration without impacting BV controls. Proliferation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) further confirmed BC1471 induced ​​selective growth arrest in STAMBP overexpressing cells​​. Collectively, these results demonstrate that pharmacological inhibition of STAMBP with BC1471 restores K63-linked ubiquitination of E2F1, thereby re-establishing RB1-imposed repression of E2F1 activity and thereby suppresses the proliferation and migration of bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDepletion of STAMBP Suppresses Bladder Cancer Tumorigenesis In Vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the role of STAMBP in bladder cancer tumorigenesis \u003cem\u003ein vivo\u003c/em\u003e, we developed a subcutaneous xenograft model incorporating STAMBP-knockout and wild-type T24 cells. Tumors derived from STAMBP-knockout cells exhibited significantly slower growth, with marked reductions in both tumor volume and weight compared with the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C), indicating that the absence of STAMBP hinders the tumorigenic potential of bladder cancer cells. Furthermore, immunohistochemical (IHC) analysis confirmed a marked decline in the number of Ki67-positive proliferating cells and levels of STAMBP protein in STAMBP- knockout tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), supporting the conclusion that STAMBP deletion diminishes tumor cell proliferation \u003cem\u003ein vivo\u003c/em\u003e. Mechanistically, western blot analysis of tumor tissues showed substantial downregulation of key E2F1 downstream targets, including CDC2 and DHFR, in STAMBP-knockout tumors relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Collectively, these findings highlight STAMBP as a critical promoter of bladder cancer tumorigenesis, whose loss impedes tumor growth by downregulating E2F1-mediated proliferative pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eStambp Deficiency Lessens BBN‑Induced Bladder Tumorigenesis in Mice\u003c/h2\u003e \u003cp\u003eTo clarify the tumorigenic role of STAMBP in bladder cancer \u003cem\u003ein vivo\u003c/em\u003e, CRISPR/Cas9 strategy was employed to generate Stambp bladder conditionally knockout mice (Stambp\u003csup\u003eflox/flox\u003c/sup\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We generated bladder-specific \u003cem\u003eSTAMBP\u003c/em\u003e knockout mice (Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre) by crossing Stambp\u003csup\u003efl/fl\u003c/sup\u003e mice with Upk2-Cre transgenic mice, which express Cre recombinase under the urothelial-specific uroplakin 2 (Upk2) promoter. Genomic PCR confirmed the successful recombination of the Stambp locus in homozygous mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). No significant differences in body weight were observed between Stambp\u003csup\u003eflox/flox\u003c/sup\u003e Upk2-Cre mice and Stambp\u003csup\u003efl/fl\u003c/sup\u003e mice, indicating that Stambp deletion did not induce systemic toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). To assess the impact of STAMBP deficiency on bladder tumorigenesis, we subjected Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre and control mice to a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced carcinogenesis model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Beginning at five weeks of age, mice received 0.05% BBN in drinking water for 21 weeks. At the 26-week endpoint, bladder tissues were harvested for analysis. Mice lacking Stambp exhibited a notable reduction in tumor burden induced by BBN when compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Bladder weights, serving as a surrogate marker of tumor progression, were significantly lower in the Stambp\u003csup\u003eflox/flox\u003c/sup\u003e; Upk2-Cre mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Histopathological evaluation revealed that 50% of tumors in knockout mice were in the early stages (Stage I), while 83% of tumors in the control group had progressed to more advanced stages (Stage II/III) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Notably, STAMBP-deficient mice displayed significantly improved survival compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Additionally, immunohistochemical (IHC) analysis highlighted a reduction in pathological damage in bladders lacking Stambp, with features such as diminished stromal invasion and maintained urothelial structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). Collectively, these findings demonstrate that the urothelial-specific deletion of STAMBP mitigates BBN-driven bladder tumorigenesis, delays tumor progression, and improves survival. underscoring STAMBP as a critical mediator of bladder cancer pathogenesis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on our mechanistic insights, we propose a 'Dual-Lock' model that delineates two distinct pathways regulating RB1-E2F1 complex stability and E2F1 activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e): (i) the canonical phosphorylation-dependent pathway, where CDK4/6-mediated RB1 phosphorylation induces conformational change-mediated release of E2F1 from the repressive complex (RB1-E2F1-HDAC1), activating cell cycle targets (e.g., CCNE1, DHFR); and (ii) a deubiquitination-driven pathway, wherein K63-linked ubiquitin chains on E2F1 (K161/164) act as molecular scaffolds that reinforce complex stability. STAMBP\u0026mdash;identified as the key K63-specific deubiquitinase\u0026mdash;removes these chains, dismantling the scaffold and destabilizing the complex without affecting RB1 phosphorylation; this liberates transcriptionally active E2F1 to drive malignant progression. Together, the model provides a mechanistic framework for understanding context-specific E2F1 activation in bladder cancer pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBladder cancer represents a major global health burden. In 2025, it is projected that 17,420 deaths in the United States will be attributable to bladder cancer, representing the highest mortality among urological malignancies in men(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Bladder cancer develops through two distinct pathways, resulting in non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Standard therapies for NMIBC involves transurethral resection of the bladder tumor (TURBT) followed by intravesical Bacillus Calmette-Gu\u0026eacute;rin (BCG), however, BCG failure occurs in approximately 40% of patients with high-risk disease​(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). For MIBC, cisplatin-based neoadjuvant chemotherapy (NAC) remains the standard of care, yet up to 50% of patients with renal or cardiac comorbidities are ineligible(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)​​. These limitations underscore the urgent need for precision-based therapeutic approaches that can enhance treatment efficacy. Encouragingly, recent advances in molecular subtyping of bladder cancer have facilitated the development of targeted therapies, such as FGFR3 inhibitors, as transformative treatments for this disease(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Beyond FGFR3 inhibition, multidimensional targeting strategies encompassing Nectin-4-directed agents, immune checkpoint blockade (PD-1/PD-L1), HER2 pathway modulation, tumors with DNA repair deficiencies such as those harboring ERCC2 mutations, and novel epigenetic targets (e.g., BLM lactylation) have emerged as pivotal therapeutic avenues(\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Nevertheless, these strategies do not address all patient populations, highlighting the continued need to identify additional therapeutic targets.\u003c/p\u003e \u003cp\u003eAs the important components of post translational modification, ubiquitin and de-ubiquitin family enzymes are involved in the initiation and progression of various diseases, including multiple cancers(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Modulating DUB activity has emerged as a promising therapeutic strategy to suppress tumor proliferation and improve responses to chemotherapy and immunotherapy(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Recent studies indicate several deubiquitinases, including BRCC3, OTUB1, and UCHL3, are aberrantly expressed in bladder cancer and play functional roles in its development(\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In the present research, we identified STAMBP as another critical DUB gene associated with bladder cancer proliferation. By validating databases and tissue specimens, we demonstrate that STAMBP is highly expressed in bladder cancer. We conducted functional experiments and demonstrated that increasing levels of STAMBP facilitated the growth and invasion of BC cells, whereas the knockout of STAMBP hindered the aggressive phenotype of BC cells. Consistent with its oncogenic role, pharmacological inhibition of STAMBP by BC1471 (10 \u0026micro;M) significantly suppressed proliferation and migration in the STAMBP-high BC cells compared with control cells. Earlier research indicates that STAMBP enhances the migration and invasion of lung cancer cells and triple-negative breast cancer (TNBC) cells(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Consistent with these investigations, our results suggest that STAMBP represents a potential therapeutic target in bladder cancer.\u003c/p\u003e \u003cp\u003eRNA sequencing combined with Gene Ontology (GO) enrichment analysis revealed a strong association between STAMBP and cell cycle regulation. Further analysis reveals a strong association between STAMBP and the E2F signaling pathway; this result has been validated through dual-luciferase reporter gene experiments. Members of the E2F signaling pathway act as transcription factors forming the central transcription axis(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Real-time quantitative PCR showed that STAMBP depletion significantly downregulates CDCA2, DHFR, and PCNA expression. The results from protein-level assays indicate that the loss of STAMBP leads to a decrease in the protein levels of CDCA2 and DHFR, while graded expression of STAMBP results in a dose-dependent increase in their protein levels. Together, these findings suggest that STAMBP positively regulates the transcriptional activator E2F1 within the E2F family. previous studies reported that elevated E2F1 expression in bladder cancer promotes progression from superficial to invasive stages(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, we primarily focus on the molecular mechanisms of E2F1 in our studies.\u003c/p\u003e \u003cp\u003eThe RB1\u0026ndash;E2F1 axis represents the canonical model of E2F1 regulation. In this model, phosphorylation induces conformational changes in RB1 under specific conditions, releasing E2F1 to activate transcription of downstream genes(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Consequently, numerous studies have focused on the changes in the phosphorylation state of RB1. Additionally, many studies have concentrated on the post-translational modifications of E2F1, including phosphorylation, methylation, and acetylation(\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Ubiquitination and deubiquitination, which are critical for protein homeostasis, also play important roles in E2F1 regulation, particularly through K63-linked ubiquitination. UCH37 is the first reported deubiquitinating enzyme capable of removing K63-linked ubiquitination from E2F1. Although this does not alter E2F1 protein stability, it enhances transcriptional activity through deubiquitination(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). POH1 stabilizes E2F1 by removing both K63- and K11-linked ubiquitin chains, thereby preventing proteasomal degradation(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). STAMBP has also been shown to directly bind to E2F1 and stabilize its expression in pancreatic cancer cells, possibly by inhibiting K48-linked polyubiquitination; however, this mechanism requires further experimental validation(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Despite these insights, it remains unclear whether ubiquitination enhances E2F1 stability, how this is related to RB1 phosphorylation, and whether it affects RB1-E2F1 binding. Our research demonstrates that STAMBP enhances the stability of E2F1. Laurence Dubrez et al., reported that lysine residues 161 and 164 of E2F1 are critical for its K63 polyubiquitination modification(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Therefore, we evaluated the effect of STAMBP on K63 ubiquitination of the E2F1 K161/164R mutant. The results showed that STAMBP treatment did not alter the K63 ubiquitination level of the E2F1 K161/164 mutant, indicating that STAMBP removes K63-linked ubiquitin chains to deubiquitinate E2F1. We also found that the addition of STAMBP weakens the interactions between RB1 and E2F1. We also examined the phosphorylation levels of RB1. We found that STAMBP does not affect RB1 phosphorylation. This observation raises an important question: if STAMBP does not influence RB1 phosphorylation, why is the interaction between E2F1 and RB1 reduced? Given that K63-linked ubiquitin chains act as scaffolds for protein-protein interactions, we propose a model where RB1-E2F1 binding is regulated by both RB1 pocket conformation and K63 polyubiquitination. K63 polyubiquitination reinforces the RB1-E2F1 interaction, whereas STAMBP-mediated removal of these chains disrupts binding, releasing E2F1. This release enhances downstream survival signaling, promoting the occurrence of bladder cancer.\u003c/p\u003e \u003cp\u003eTo further explore the potential of STAMBP as a therapeutic target, we conducted in \u003cem\u003evivo\u003c/em\u003e experiments. We first verified that the deletion of STAMBP inhibited the tumor-forming ability of bladder cancer cells in vivo using a nude mouse tumor model. In addition, we generated bladder-specific STAMBP knockout mice and employed a BBN-induced bladder carcinogenesis model. The results showed that the deletion of STAMBP increased the survival time of mice in the BBN-induced tumor model, while also reducing tumor volume and decreasing tumor progression severity.\u003c/p\u003e \u003cp\u003eTranscription factors like E2F1 are appealing therapeutic targets. However, their broad functional pleiotropy and nuclear localization make tumor-specific inhibition particularly challenging. DUBs have recently emerged as promising druggable targets in oncology, with several inhibitors (e.g., PR-619 against USP14) currently undergoing clinical evaluation(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Moreover, Our study establish STAMBP as a pivotal oncogenic DUB in bladder cancer. We demonstrate that STAMBP destabilizes the RB1-E2F1 transcriptional repressor complex via K63-linked deubiquitination, thereby promoting cell-cycle progression and metastatic phenotypes. These findings provide a strong therapeutic rationale for targeting STAMBP. However, limitations remain. Current tissue analyses lack subtype-specific stratification and validation across multicenter cohorts. Additionally, translational challenges include optimizing BC1471's therapeutic window and pharmacodynamics in patient-derived xenograft models before clinical assessment. Future studies should also correlate STAMBP expression with chemotherapy response in Phase II trials, which may accelerate its development as a predictive biomarker.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study uncovers a phosphorylation-independent regulatory axis governing the RB1-E2F1 complex, mediated by the deubiquitinase STAMBP. We demonstrate that RB1-E2F1 complex integrity is co-regulated by classical phosphorylation mediates RB1 \"pocket\" conformation and K63-ubiquitin \"Scaffold\" of E2F1. Critically, we identify STAMBP as an oncogenic trigger and a therapeutic target for a molecularly defined subset of BC patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAMSH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAssociated molecule with the SH3 domain of STAM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBBN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eN-butyl-N-(4-hydroxybutyl)-nitrosamine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBladder cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eBCG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eBacillus Calmette–Guérin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCDK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCyclin-dependent kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCDC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCell division control protein 2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3,3′-Diaminobenzidine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDUB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDeubiquitinating enzyme\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eE2F1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eE2F transcription factor 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEGFR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEpidermal growth factor receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eEMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eEpithelial–mesenchymal transition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eFGFR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eFibroblast growth factor receptor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHistone acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eHDAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eHistone deacetylase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eJAMM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eJAB1/MPN/Mov34 metalloenzyme\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eMIBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMuscle-invasive bladder cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNeoadjuvant chemotherapy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eNMIBC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eNon-muscle-invasive bladder cancer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePKA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eProtein kinase A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eRB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eRetinoblastoma 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSTAMBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eSTAM-binding protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTissue microarray\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTURBT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eTransurethral resection of bladder tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professor Tian-Zhi Huang from Xiamen university and Will Fong from University of Kentucky for their numerous suggestions throughout the paper writing process. The schematic diagram (Fig.6J) was created using Figdraw.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by grants from the National Natural Science Foundation of China (32270760), the Fundamental Research Funds for the National Key R\u0026amp;D Project (2022YFF0710700).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. Liu: Project administration, investigation, data curation, validation, visualization. QP. Shu: Investigation, supervision, validation, visualization. L. Yu: Investigation,\u0026nbsp;validation, writing–original draft. CM. Jiang: Investigation, formal analysis, visualization. HH Tao: Investigation, formal analysis, software, Methodology. WZ Wang: Investigation, visualization. J Zhang: Investigation, data curation. YF Pang: Data curation, software. C. M: Formal analysis, data curation, supervision. HH. Zhang: Data curation, supervision, writing–review and editing. SZ. Li: Conceptualization, resources, funding acquisition, project administration, writing–review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Shang-Ze Li, Hui-Hui Zhang, and Chao Ma.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in compliance the Guidelines of the China Animal Welfare Legislation and were approved by the Committee on Ethics in the Care and Use of Laboratory Animals of Wuhan University (permit number: WP20230008, WP2020-08018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. 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Immunology \u003cstrong\u003e2023\u003c/strong\u003e;170:439-51\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"STAMBP, E2F1, deubiquitination, bladder cancer, RB1 ","lastPublishedDoi":"10.21203/rs.3.rs-8327775/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8327775/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe canonical RB1-E2F regulatory model depends on RB phosphorylation-induced allosteric changes during the cell cycle. However, extensive RB mutations across cancers indicate the existence of phosphorylation-independent mechanisms governing RB\u0026ndash;E2F complex stability. Here, we report a novel regulatory axis where enhanced E2F1 activity inversely correlates with K63-linked ubiquitination levels, independent of changes in RB1 phosphorylation status. Through systematic deubiquitinase profiling, we identify STAM Binding Protein (STAMBP)\u0026mdash;a K63-specific deubiquitinase overexpressed in bladder tumors and correlated with advanced disease and poor survival\u0026mdash;as the key enzymatic regulator. Mechanistically, STAMBP binds E2F1 and removes K63 chains at lysines 161/164, destabilizing the RB1-E2F1 repressive complex while maintaining RB1 phosphorylation homeostasis. This enhances E2F1 transcriptional activity, driving cell cycle target gene expression and promoting malignant progression through proliferation and invasion. Genetic loss of STAMBP suppresses tumor growth in vitro and in vivo. Bladder-specific Stambp knockout delays carcinogen-induced tumor progression and improves survival, while pharmacological inhibition with BC1471 selectively blocks proliferation in STAMBP-high cells without toxicity. Together, these findings establish a 'Dual-Lock' paradigm: K63-linked ubiquitin chains act as a molecular scaffold stabilizing the RB1-E2F1-HDAC1 complex, whereas STAMBP-mediated deubiquitination triggers oncogenic E2F1 activation. This work nominates STAMBP as a biomarker-driven therapeutic target for precision oncology in bladder cancer.\u003c/p\u003e","manuscriptTitle":"STAMBP-Mediated K63 Deubiquitination of E2F1 Release E2F1 from RB Repressive Complex to Drive Bladder Cancer Progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 14:44:28","doi":"10.21203/rs.3.rs-8327775/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"33ad0b00-a5bb-4e24-a000-57d5b8feb198","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59682374,"name":"Biological sciences/Cancer/Oncogenes"},{"id":59682375,"name":"Health sciences/Diseases/Urogenital diseases"}],"tags":[],"updatedAt":"2026-01-09T17:51:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 14:44:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8327775","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8327775","identity":"rs-8327775","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}