OTUD4 suppresses colorectal cancer progression through deubiquitinating p53

OTUD4 suppresses colorectal cancer progression through deubiquitinating p53 | 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 Research Article OTUD4 suppresses colorectal cancer progression through deubiquitinating p53 Xianyue Bu, Yingxu Wang, Jinzhen Pan, Shuai Li, Penghao Li, Bing Zheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7187190/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Colorectal cancer (CRC) represents a global health burden as the third most commonly diagnosed malignancy and the second leading cause of cancer-related mortality. Despite advances in research, molecular mechanisms driving CRC initiation, progression, and metastasis remain incompletely elucidated. OTU domain-containing protein 4 (OTUD4), a deubiquitinating enzyme of the ovarian tumor family, catalyzes ubiquitin chain removal from substrate proteins. While implicated in diverse cellular processes, OTUD4's role in CRC pathogenesis is undefined. Here, we report significant downregulation of OTUD4 in CRC specimens relative to normal colonic epithelium, and demonstrate OTUD4 depletion enhances proliferation, clonogenicity, migration, and invasion in CRC cell lines, consistent with tumor-suppressive activity. Mechanistically, OTUD4 interacts with and deubiquitinates p53, thereby stabilizing this tumor suppressor protein and enhancing its transcriptional activity. Critically, p53 knockdown abrogates OTUD4-mediated suppression of malignant phenotypes, establishing the OTUD4-p53 axis as a critical regulatory node in CRC. Our findings identify OTUD4 as a novel tumor suppressor that constrains colorectal carcinogenesis through deubiquitinating and stabilizing p53, highlighting its therapeutic potential and warranting deeper investigation of OTUD4 in cancer biology. CRC OTUD4 p53 Cancer progression Deubiquitination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Colorectal cancer (CRC) is the third most prevalent diagnosed malignancy and a leading cause of cancer-related mortality worldwide 1 . Despite advancements in surgical techniques and chemotherapeutic regimens, patient outcomes remain suboptimal due to high rates of metastasis, recurrence, and treatment resistance 2 . These challenges are compounded by the complex molecular landscape of CRC, which involves a multitude of genetic and proteomic alterations contributing to tumor initiation, progression, and relapse 3 . Key genetic mutations frequently observed in CRC include alterations in TP53, KRAS, and APC genes, as well as deficiencies in DNA mismatch repair mechanisms, leading to microsatellite instability 4 , 5 . However, the precise molecular mechanisms driving CRC pathogenesis are not fully elucidated. The p53 transcription factor acts as an indispensable guardian against cancer, modulating a broad spectrum of cellular activities crucial for genomic integrity, including DNA damage repair, progression of cell cycle, induction of senescence, initiation of cell death pathways, facilitation of differentiation, and regulation of metabolic processes 6 , 7 . p53 and the related signaling is frequently dysregulated in colorectal cancer, leading to tumorigenesis and progression 8 – 10 . Other than missense mutation and transcription dysregulation, post-translational modifications, such as ubiquitination, phosphorylation, and acetylation, critically regulate the function of p53 in CRC 11 – 13 . Ubiquitination constitutes a pivotal post-translational modification event for p53 regulation. MDM2, an established ubiquitin ligase, promotes K48-linked polyubiquitination of p53, targeting it for subsequent degradation. 14 , 15 . Besides, other E3 ligases such as COP1, UFL1, and FBXL6 also modulate p53 ubiquitination 14 , 16 . However, the precise molecular mechanisms governing p53 ubiquitination which involving in CRC progression remain incompletely understood and warrant further elucidation. OTU domain-containing protein 4 (OTUD4) is a deubiquitinating enzyme (DUB) belonging to the ovarian tumor (OTU) family, capable of hydrolyzing Lys48- and Lys63-linked polyubiquitin chains from modified proteins 17 , 18 . OTUD4 participates in diverse biological processes, including innate immune response 17 , antiviral reaction 19 , DNA damage and RNA binding 20 . In cancer, OTUD4 has been reported to promote glioblastoma 21 , breast cancer 22 , 23 , melanoma 24 progression via its deubiquitinating activity. Conversely, other studies indicated that OTUD4 acts as a tumor suppressor in intrahepatic cholangiocarcinoma 25 , non-small cell lung cancer 26 , ovarian tumor 27 . The context-dependent role of OTUD4 in tumor progression, whether oncogenic or tumor-suppressive, remains to be fully elucidated. In this study, we demonstrated the lower expression of OTUD4 in CRC tissues compared to adjacent normal tissues. OTUD4 suppresses CRC cells progression in vitro and in vivo by deubiquitinating and stabilizing tumor suppressor p53. Collectively, our findings reveal a novel tumor-suppressive mechanism of OTUD4 by stabilizing p53, thereby inhibiting colorectal cancer development. Methods and materials Cell lines and culture conditions The human CRC cell line HCT116, murine CRC cell line CT26 and Human Embryonic Kidney 293T cells were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). All the cell lines and were cultured at 37℃ with 5% CO 2 in in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Routine mycoplasma testing confirmed all lines were free of contamination. Patients and tissue specimens Tumor tissue microarrays of CRC patients were obtained from Department of Anorectal Surgery, the Second Hospital of Tianjin Medical University. All the samples and follow-up data acquisition were under the approval of Human Ethics Committee of Second Hospital of Tianjin Medical University (Approval Number: No.KY2023K207). Written informed consent was obtained from all the patients. All the methods were carried out in accordance with relevant guidelines. IHC Analysis Antibody detection was performed using standard IHC methods and the result of staining was evaluated using a semi-quantitative scoring system as previously described. The staining intensity (0: negative; 1: weak; 2: moderate; 3: strong) and percentage of positively stained cells (0: 75%) were determined independently. A composite IHC score (range 0–12) was calculated by multiplying the intensity score by the frequency score. The detailed information of antibodies is listed in Supplementary Table S1 . Plasmids and shRNA Full-length human OTUD4 cDNA (CCDS47139.1) and murine Otud4 (CCDS57625.1) was cloned into the vector with an N-terminal HA tag. Human TP53 cDNA (CCDS11118.1) was cloned into a pLenti vector with an N-terminal Flag tag. pLKO.1 vectors carrying shRNA targeting OTUD4 and p53 was purchased from Tsingke Biotechnology Company (Beijing, China). Indicated sequence of shRNA target are listed in Supplementary Table S2 . Lentiviral Production and Transduction Lentiviral particles were produced in 293T cells via PEI-mediated transfection (40816ES02, Yeasen). Cells were transfected with 2 µg of lentiviral plasmid, 1.5 µg psPAX2, and 0.5 µg pMD2.0 G. Viral supernatant was harvested 48h and 72h post-transfection, pooled, filtered (0.45 µm), and used to infect CRC cells. Stable cell lines were selected using 1–5 µg/mL puromycin for at least two weeks. Immunoprecipitation (IP) For Co-IP, cells were washed with ice-cold PBS, harvested, and lysed using IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05 mM EDTA, 1% NP-40, 20% glycerol) supplemented with 1% protease/phosphatase inhibitor cocktail (Transgen, DI111-01). Lysate was clarified by centrifugation at 20,000g for 5 min at 4℃. Target proteins were then immunoprecipitated by incubating the primary bodies and pre-cleared protein-G magnetic beads at 4℃ overnight. After extensive washing with IP buffer, immunoprecipitated complexes were eluted for Western blotting or mass spectrometry (MS) analysis. Western blotting Cells were lysed with denaturing buffer (1% SDS, 50 mM Tris-HCl Ph 7.4 and 2 mM EDTA in ddH2O) and then quantified by nanodrop after boiled for 10 min at 100℃. Proteins (40 µg per lane) were separated on SDS-PAGE, transferred to NC membranes (Millipore), blocked by 5% no-fat milk, and then incubated with primary antibodies overnight at 4℃. Following three washes, the membranes were incubated with HRP-conjugated secondary antibody and detected using enhanced chemiluminescence (ECL). Housekeep gene GAPDH as used as a loading control. The detailed information of antibodies is listed in Supplementary Table S1 . In vivo ubiquitination assays In vivo ubiquitination assays were performed as previously described. In brief, 293T cells were co-transfected with His-Ub, HA-OTUD4 and Flag-p53 plasmids. cells were treated with 10 µM MG-132 (Sigma-Aldrich, 133407-82-6) for 4 h to inhibit proteasomal degradation. Cells were harvested and lysed in denaturing buffer (6 M guanidine-HCl, 100 mM Na₂HPO₄/NaH₂PO₄, 10 mM Tris-HCl, 5 mM imidazole, pH 8.0). Ubiquitinated proteins were enriched under denaturing conditions using Ni-NTA agarose beads (QIAGEN, 30210) to isolate His-ubiquitin conjugates. Ubiquitination of Flag-p53 was detected by immunoblotting with anti-Flag antibody. Reverse transcription – quantitative PCR (RT – qPCR) For RT–qPCR, total RNA was extracted using Trizol Reagent (Invitrogen) and was reverse transcripted to cDNA by HiScript II Reverse Transcriptase kit (Vazyme, R211). SYBR Green Supermix (Vazyme, Q712) was used to detect the corresponding mRNA level using and GAPDH was used as an internal control. All the sequence of primers used in this article were listed in Table S3 . Cell cycle and apoptosis analysis For cell cycle analysis, cells were collected after twice washing by cold PBS and fixed with 70% ethanol overnight at 4℃. Then cells were resuspended with RNase A and propidium iodide (PI, Beyotime, Y267501) for 20 min at room temperature and then analyzed by detecting PerCP 5.5 using flow cytometry. For apoptosis analysis, cells were collected with trypsin without EDTA and then stained with Annexin V and PI. The cells were analyzed by detecting FITC and PerCP 5.5 using flow cytometry. CCK-8 assays, Colony formation and Transwell assays For CCK-8 assays, 1000 cells were seeded into 96-well plates with 100 µl complete DMEM medium. The cell numbers were counted by CCK-8 reagent (Beyotime, C0037) using a microplate spectrophotometer at different points in time. Absorbance at 450 nm was employed to determine the number of viable cells and cell viability was calculated based on a standard curve. For colony formation assay, 300 cells were seeded in 6-well plates with 3 mL complete DMEM medium. After 10–14 days, colonies were washed with PBS, fixed with 100% methanol for 15 min, and stained with 0.5% crystal violet for 30 min at room temperature. Crystal violet (Beyotime, Y268091) stained colonies (> 50 cells/colony) were automatically quantified using ImageJ v1.53. For Transwell assays, Cell migration was assessed using 8-µm pore Transwell inserts (Corning). 5 × 10⁴ cells in 200 µL serum-free DMEM were seeded into the upper chamber. The lower chamber contained 500 µL DMEM with 20% FBS. After 24 h incubation, migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted in 5 random fields per insert (100 × magnification). Animal study Tumor xenograft studies were approved by the Institutional Animal Care and Use Committee, and animal experiments were performed in accordance with protocols approved by the Second Hospital of Tianjin Medical University Animal Care and Use Committee. (No. TMU2024119). The 6-week-old male nude mice were purchased from Cyagen Biosciences (Guangzhou, China). For experiments, 5 mice per group would be sufficient to detect a difference based on previous experience with the model, and animals were randomly assigned to different groups. Investigators were not blinded to group allocation. SPF mice were maintained under specific pathogen-free conditions with controlled temperature (22–24°C), humidity (40–70%), sterile feed/water, and IVC housing, following institutional animal care protocols. CT26 cells expressing vector control or OTUD4 (1×10 6 cells in 100 µL PBS) were injected subcutaneously into the right flank of mice after being anesthetized via intraperitoneal injection of tert-amyl alcohol/tribromoethanol. Tumor volume was measured every 3 days using digital calipers from day 7 post-injection until day 16, calculated as: Tumor volume (mm 3 ) = 0.5 × Length × Width 2 . On day 16, mice were euthanized by CO₂ asphyxiation followed by cervical dislocation, and tumors were excised. The maximal tumor size burden (20% in any dimension or 10% of body weight) was not exceeded, which was permitted by the ethics committee. All the tumor size in animals is less than 500 mm³, which generally does not cause pain, weight loss, or physiological distress in mice. Quantification and statistical analysis All the ex vivo experiments were repeated at least three times and data are presented as the mean ± SD. The data presentation and statistical analyses are described in the figure legends. Differences with P < 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism 9.0 Software ( https://www.graphpad.com/features ). Data availability The cDNAs used during the current study are available in the PubMed repository (human OTUD4 : CCDS47139.1; Murine Otud4 : CCDS57625.1; Human TP53 : CCDS11118.1.), [ https://www.ncbi.nlm.nih.gov/gene ]. Result Lower OTUD4 Expression Correlates with Poor Prognosis in Colorectal Cancer To investigate the dysregulation of OTUD4 in colorectal cancer (CRC), we first assessed its mRNA expression using the GEPIA database ( http://gepia.cancer-pku.cn/ ) and found that both colon and rectal carcinoma tissues exhibited significantly lower OTUD4 mRNA levels compared to matched normal tissues (Fig. 1 A). Confirming these findings at the protein level, proteomic analysis of the International Cancer Proteogenome Consortium (ICPC) datasets revealed a marked reduction of OTUD4 in tumor tissues relative to adjacent normal counterparts (Fig. 1 B). Furthermore, IHC staining of tissue microarrays (TMAs) from our clinical cohort revealed notably diminished OTUD4 protein expression in CRC samples compared to that in normal (Fig. 1 C). We next examined the prognostic implications of reduced OTUD4 expression. We found that CRC patients with lower OTUD4 expression had a shorter relapse-free survival (RFS) and overall survival (OS) using the TCGA dataset via the Kaplan–Meier Plotter platform [CRC] ( https://kmplot.com/analysis ) and GEPIA data ( http://gepia.cancer-pku.cn/detail.php?gene=OTUD4 ) (Fig. 1 D and E). Together, these results suggest that OTUD4 is downregulated in CRC and is associated with poorer prognosis. OTUD4 Suppresses CRC Cell Proliferation, Promotes Apoptosis, and Inhibits Migration To investigate the function of OTUD4 in CRC cells, the OTUD4 knockdown and control HCT116 cells were constructed using lentiviral. The cell cycle assay results revealed that OTUD4 knockdown significantly decreased the proportion of cells in G2/M phase (Fig. 2 A). Correspondingly, Annexin V assays demonstrated a marked decrease in apoptotic cell population upon OTUD4 knockdown (Fig. 2 B). Furthermore, CCK8 and clonal formation assays demonstrated that OTUD4 negatively regulated the growth of HCT116 cells (Fig. 2 C and D). Additionally, Transwell assay showed that knockdown of OTUD4 enhanced the migration ability of HCT116 cells (Fig. 2 E). Collectively, these data indicate that OTUD4 acts as a tumor suppressor by restraining cell cycle progression, enhancing apoptosis, and inhibiting proliferation and migration in CRC cells. OTUD4 Deubiquitinates and Stabilizes p53 to in CRC To elucidate the mechanism by which OTUD4 suppresses CRC, we overexpressed HAtagged OTUD4 in HCT116 cells, performed immunoprecipitation, and identified interacting substrates via mass spectrometry. Notably, the tumor suppressor p53 emerged among OTUD4-binding proteins (Fig. 2 A). Accordingly, we hypothesized that OTUD4 stabilizes p53 through its deubiquitinase activity. Endogenous OTUD4–p53 interaction was confirmed in wildtype p53 HCT116 cells (Fig. 2 B). Subsequent in vivo ubiquitylation assays revealed that OTUD4 overexpression markedly diminished polyubiquitylation of p53 (Fig. 2 C). To determine whether OTUD4 influences p53 protein stability, we overexpressed OTUD4 in CT26 cells, observing increased p53 levels, while OTUD4 knockdown reduced p53 expression (Fig. 2 D). Consistently, OTUD4 overexpression substantially extended p53 half-life in CT26 cells (Fig. 2 E). Further, OTUD4 depletion in HCT116 cells led to decreased levels of cleaved caspase‑3, while OTUD4 overexpression enhanced caspase‑3 cleavage. Correspondingly, both mRNA and protein levels of the canonical p53 target gene p21 increased following OTUD4 overexpression—despite p53 mRNA remaining unchanged (Fig. 2 F–I). Crucially, shRNA-mediated p53 knockdown abolished OTUD4-induced upregulation of cleaved caspase‑3 and p21, thereby establishing the dependence on p53. Collectively, these data show that OTUD4 directly deubiquitinates p53, enhancing its protein stability and triggering p53-dependent transcriptional activation of apoptosis and cell-cycle arrest pathways. OTUD4 Suppresses CRC Cell Aggressiveness in a p53-Dependent Manner Next, we constructed OTUD4-overexpressing, OTUD4-overexpressing with p53-knockdown and corresponding control CT26 cells. As expected, overexpression of OTUD4 decreased the portion of cells in G2/M phase and decreased the portion of annexin V-positive apoptotic cells, while knockdown of p53 reversed the effects (Fig. 4 A and B). Functionally, overexpression of OTUD4 inhibited CT26 cell proliferation and migration; these suppressive effects were again abrogated by p53 knockdown (Fig. 4 C–E). Taken together, our data demonstrate that OTUD4 attenuates the aggressiveness of colorectal cancer cells in a p53-dependent manner—reinforcing the critical role of the OTUD4–p53 axis in CRC suppression. Overexpression of OTUD4 inhibited the growth of xenograft tumors in mice. To assess the in vivo effects of OTUD4 on colorectal cancer, we established subcutaneous xenografts in NOD-SCID mice using CT26 cells stably overexpress OTUD4 or empty vector control. Tumor volumes were measured every three days using calipers. Tumors derived from OTUD4 OE cells exhibited significantly reduced growth compared to control tumors (Fig. 4 A–C). For the further, IHC assays on xenografts tumors revealed elevated p53 protein levels in OTUD4 OE tumors relative to vector controls. Concurrently, proliferation marker Ki‑67 was diminished, and apoptosis marker Cleaved Caspase-3 was markedly increased (Fig. 4 C). These results confirm that OTUD4 overexpression suppresses colorectal tumor growth in vivo, likely through stabilization of p53 leading to reduced proliferation and increased apoptosis, consistent with its function as a tumor suppressor in CRC. Discussion In this study, we comprehensively investigated OTUD4 in CRC and elucidated its crucial role as a tumor suppressor. Our findings reveal that OTUD4 is downregulated at both mRNA and protein levels in CRC tissues compared to normal colon and rectal tissues, with lower expression correlated with significantly worse relapse-free and overall survival. Functional assays demonstrated OTUD4’s suppression of proliferation, cell cycle progression, migration, and its promotion of apoptosis in vitro, as well as its inhibition of tumor growth in vivo. Mechanistically, OTUD4 directly binds p53, reduces its ubiquitination, stabilizes p53, and enhances p53-dependent transcription leading to cell-cycle arrest and apoptosis. Importantly, OTUD4’s anti-tumor functions are p53-dependent (Fig. 6 ). P53 is one of the most important tumor suppressors in CRC. The CDKN1A gene, coding for the cyclin-dependent kinase inhibitor p21, was the first discovered transcriptional target of p53. Other than the suppression function of transformation and proliferation by direct inhibiting CDKs, p53-p21 regulatory axis also promotes metastasis of CRC cells via EMT promoting 28 – 30 . On the other side, p53 promotes CRC cells apoptosis by activating Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP activation and Bcl2 deactivation, thereby inhibiting tumor proliferation and promoting apoptosis 31 . We found that OTUD4 promotes the activation of cleaved caspase-3 as well as the transcription of CDKN1A (p21) by increasing the p53 protein level. These results position OTUD4 as a novel DUB that reinforces tumor suppressor pathways via p53 stabilization in CRC. This mechanism mirrors other OTU-family DUBs, such as OTUD1 32 , OTUD3 33 and OTUD5 34 , modulating of p53 activity, and is consistent with previous evidence that many DUBs regulate cell proliferation, cycle control, and apoptosis in cancer progression 35 , 36 . Our work corroborates broader findings that deubiquitination plays a vital role in maintaining protein homeostasis in CRC. Notably, OTUD4’s function in CRC appears to diverge from its role in other cancers. For instance, deubiquitylation of CD73 by OTUD4 results in CD73 stabilization and inhibit tumor immune responses of triple negative breast cancer 23 . In various type of tumors, OTUD4 not only sustains protein stability by directly deubiquitinating GPX4 but also impedes its degradation via RHEB-mediated autophagy, collectively stalling the ferroptosis pathway 37 . These findings highlight the context-dependent specificity of OTUD4 substrates across tissues. In CRC, OTUD4 appears to function primarily through the p53 axis, reinforcing p53’s stability and activity—a central tumor suppressive pathway commonly inactivated in CRC. Moreover, our in vivo data further support OTUD4’s biological relevance. In CT26 xenografts, OTUD4 overexpression decreased tumor growth and proliferation (Ki-67 expression), while increasing p53 levels and apoptosis (cleaved caspase-3). These in vivo phenotypes underscore OTUD4's potential clinical importance. From a translational perspective, our findings indicate that OTUD4 expression or activity could serve as a prognostic biomarker or therapeutic entry point. Given that DUB-targeted drugs have been explored in oncology pharmacological activation 38 or gene delivery of OTUD4 might represent a strategy to reactivate p53 signaling in CRC with intact p53. Conversely, in tumors where OTUD4 supports oncogenic pathways, targeted inhibition may be beneficial. Thus, elucidating OTUD4’s substrate specificity and regulation in CRC could guide tailored therapeutic applications. Declarations Conflict of Interest: The authors declare no conflict of interest. Ethics statement Tumor tissue microarrays of CRC patients were obtained from Department of Anorectal Surgery, the Second Hospital of Tianjin Medical University. All the samples and follow-up data acquisition were under the approval of Human Ethics Committee of Second Hospital of Tianjin Medical University (Approval Number: No.KY2023K207). Written informed consent was obtained from all the patients. All methods were carried out in accordance with relevant guidelines. Tumor xenograft studies were approved by the Institutional Animal Care and Use Committee, and animal experiments were performed in accordance with protocols approved by the Second Hospital of Tianjin Medical University Animal Care and Use Committee. (No. TMU2024119). The maximal tumor size burden (20% in any dimension or 10% of body weight) was not exceeded, which was permitted by the ethics committee. All the tumor size in animals is less than 500 mm³, which generally does not cause pain, weight loss, or physiological distress in mice. Consent to publish Not applicable. Conflict of Interest The authors confirm that this investigation was executed without any commercial or financial associations that might represent a potential conflict of interest. Funding No funding to disclose. Author Contribution XYB, YXW, JZP and BZ wrote the main manuscript text ; XYB, YXW and JZP prepared figures 1-5, SL and PHL prepared figure 6. All authors reviewed the manuscript. Acknowledgments Not applicable. References Baidoun F, et al. Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets. 2021;22:998–1009. 10.2174/1389450121999201117115717 . Eng C, et al. A comprehensive framework for early-onset colorectal cancer research. Lancet Oncol. 2022;23:e116–28. 10.1016/S1470-2045(21)00588-X . Shin AE, Giancotti FG, Rustgi AK. Metastatic colorectal cancer: mechanisms and emerging therapeutics. Trends Pharmacol Sci. 2023;44:222–36. 10.1016/j.tips.2023.01.003 . Li Q, et al. Signaling pathways involved in colorectal cancer: pathogenesis and targeted therapy. Signal Transduct Target Ther. 2024;9:266. 10.1038/s41392-024-01953-7 . Saeed M, et al. Microbe-based therapies for colorectal cancer: Advantages and limitations. 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Supplementary Files Supplementarytable12025.06.13.docx Supplementarytable22025.06.13.docx Supplementarytable32025.06.13.docx SourcedWBdata2025.08.03.pptx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Sep, 2025 Reviews received at journal 26 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviews received at journal 08 Sep, 2025 Reviewers agreed at journal 28 Aug, 2025 Reviewers invited by journal 21 Aug, 2025 Editor assigned by journal 21 Aug, 2025 Editor invited by journal 14 Aug, 2025 Submission checks completed at journal 08 Aug, 2025 First submitted to journal 08 Aug, 2025 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7187190","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506786954,"identity":"0b95bc6c-c7c7-4815-8d7e-43f3a0ada086","order_by":0,"name":"Xianyue Bu","email":"","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xianyue","middleName":"","lastName":"Bu","suffix":""},{"id":506786956,"identity":"56d18465-f957-4dbe-aada-71f05f12e8d2","order_by":1,"name":"Yingxu Wang","email":"","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingxu","middleName":"","lastName":"Wang","suffix":""},{"id":506786961,"identity":"e7f33ac1-2e22-46f4-b372-b185c3141b80","order_by":2,"name":"Jinzhen Pan","email":"","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinzhen","middleName":"","lastName":"Pan","suffix":""},{"id":506786963,"identity":"ab28365e-f3b9-48c5-a6cd-8b125a748fe4","order_by":3,"name":"Shuai Li","email":"","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Li","suffix":""},{"id":506786966,"identity":"1969568c-1338-4748-b756-9b6556101bab","order_by":4,"name":"Penghao Li","email":"","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Penghao","middleName":"","lastName":"Li","suffix":""},{"id":506786967,"identity":"13efa074-8d51-47ee-9195-8824ecc2941a","order_by":5,"name":"Bing Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBAC+/b2g4//GNjI8bM3EKnFgOdMsgFPQZqxZM8BYrVIJJhJ8Hw4nLhhRgKRWswZEpINJAyYGTdIPt54g6HGJpqgFsuGgwcfGBiwMZtLpxVbMBxLy20gqOdgQ7JBggEPm+XsHDMJxobDRGg5zGAmccBAgsfg5hkitRgcYzCTbDAAeucGD5FaJHt4ko0ZgE6T7AH6JYEYv/DLPz/4mOHP//p+9sMbb3yosSHCL8iOlEggRTlEC6k6RsEoGAWjYGQAAJTpPu2PnnDrAAAAAElFTkSuQmCC","orcid":"","institution":"the Second Hospital of Tianjin Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-07-22 12:53:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7187190/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7187190/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90197567,"identity":"2cdc10b4-98e7-4834-9937-eed07186337a","added_by":"auto","created_at":"2025-08-29 17:43:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":268012,"visible":true,"origin":"","legend":"\u003cp\u003eLower OTUD4 expression correlates with poor prognosis in CRC. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e mRNA expression and protein level of OTUD4 in CRC and normal tissues were analyzed using GEPIA (A) and ICPC (B) databases respectively. \u003cstrong\u003eC\u003c/strong\u003e Protein levels of OTUD4 in CRC and normal tissues were detected using IHC staining, representative image of tumor and normal tissue was presented, and IHC score were calculated and analyzed. D and E RFS and OS of the patients with different expression levels of OTUD4 were analyzed using Kaplan–Meier Plotter platform and GEPIA database. Data are presented as mean ± SD. Statistical analyses were performed with the unpaired Student’s t test, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/a6c6888d14b2ce451171eae0.jpg"},{"id":90197063,"identity":"f18d3bed-26a4-45f8-99f8-a83b9f60e92b","added_by":"auto","created_at":"2025-08-29 17:35:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230695,"visible":true,"origin":"","legend":"\u003cp\u003eOTUD4 suppresses CRC cells aggressive \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e Cell cycle distribution (A) and apoptosis rates (B) of the HCT116-shOTUD4 and HCT116-shControl cells were detected using flow cytometry. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003eProliferation ability of the indicated cells were detected using CCK-8 assays (C) and colonel formation assays (D). \u003cstrong\u003eE\u003c/strong\u003e Migration ability of the indicated cells were detected using Transwell assays. Statistical analyses were performed with the unpaired Student’s t-test (A, B, D and E) or two-way ANOVA (C). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Bar = 50 μm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/aeaceefaccedf6a235498367.jpg"},{"id":90198013,"identity":"921c0b5d-02d8-45d8-a21c-59389dd42ee5","added_by":"auto","created_at":"2025-08-29 17:51:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313053,"visible":true,"origin":"","legend":"\u003cp\u003eOTUD4 deubiquitinates and stabilizes p53. \u003cstrong\u003eA\u003c/strong\u003e protein interactors of OTUD4 were identified using IP and MS assay. \u003cstrong\u003eB\u003c/strong\u003e Interaction between OTUD4 and p53 proteins in HCT116 cells was detected using Co-IP and immunoblot. \u003cstrong\u003eC\u003c/strong\u003e 293T cells were transiently co-transfected with His-Ub, Flag-p53 and HA-OTUD4 or vector control for 48 h followed 4 h treatment with 10 μM MG132. Ubiquitination levels of exogenous p53 were detected using IP and immunoblot. \u003cstrong\u003eD \u003c/strong\u003eProtein levels of p53 and OTUD4 in the indicated cells were detected using WB. \u003cstrong\u003eE\u003c/strong\u003eThe impact of OTUD4 on the half-life of p53 in the CT26 cells was evaluated by WB. \u003cstrong\u003eF\u003c/strong\u003e and \u003cstrong\u003eH \u003c/strong\u003eProtein levels of CC-3 and p21 in the indicated cells were detected using WB. G and I mRNA levels of OTUD4, p53 and p21 were detected using RT-qPCR. Statistical analyses were performed with the unpaired Student’s t-test. **\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt;0.0001. CC-3, cleaved caspase-3; WCL, whole cell lysate.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/4bf817e04b6440761562df14.jpg"},{"id":90197573,"identity":"d6b8eabd-4389-4ab2-9500-c13cff469760","added_by":"auto","created_at":"2025-08-29 17:43:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":241125,"visible":true,"origin":"","legend":"\u003cp\u003eOTUD4 suppresses CRC cell aggressiveness in a p53-dependent manner. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eB\u003c/strong\u003e Cell cycle distribution (A) and apoptosis rates (B) of the CT26-Vector, CT26-OTUD4\u003csup\u003eOE\u003c/sup\u003e, and CT26-OTUD4\u003csup\u003eOE \u003c/sup\u003e+ plus shp53 cells were detected using flow cytometry. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Proliferation ability of the indicated cells were detected using CCK-8 assays (C) and colonel formation assays (D). \u003cstrong\u003eE\u003c/strong\u003e Migration ability of the indicated cells was detected using Transwell assays. Statistical analyses were performed with the unpaired Student’s t-test (A, B, D and E) or two-way ANOVA (C). *\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\u003eP\u003c/em\u003e \u0026lt; 0.0001. Bar = 50 μm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/92bb000b38abd9ad10818822.jpg"},{"id":90197077,"identity":"444c6de3-714d-4e89-b26e-e6940698ae7b","added_by":"auto","created_at":"2025-08-29 17:35:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425640,"visible":true,"origin":"","legend":"\u003cp\u003eOTUD4 inhibited the growth of xenograft tumors \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e Xenograft tumor of CT26-Vector and CT26-OTUD4\u003csup\u003eOE \u003c/sup\u003ecells were transplanted into nude mice (n = 5). Tumor volumes were measured every 3 days. D expression of OTUD4, p53, Ki67 and cleaved caspase caspase 3 in indicated tumor tissue were detected using IHC and quantified using IHC score. Statistical analyses were performed with the unpaired Student’s t-test (C and D) or two-way ANOVA (B). *\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\u003eP\u003c/em\u003e \u0026lt; 0.0001. Bar = 50 μm. CC-3, cleaved caspase-3.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/ef77b0d3d495a8bc352fa0b1.jpg"},{"id":90197080,"identity":"0af78b80-82bf-40ef-9c70-77951ff3243b","added_by":"auto","created_at":"2025-08-29 17:35:07","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":126630,"visible":true,"origin":"","legend":"\u003cp\u003eThe graphical abstract illustrating OTUD4 deubiquitinates and stabilized p53 to promote CRC cells aggressiveness.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/e106450131a009c85f727152.jpg"},{"id":91148060,"identity":"551e1be9-cc43-41a5-ba92-0107f567c347","added_by":"auto","created_at":"2025-09-12 06:41:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2432176,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/06026754-4b83-48c8-91eb-e2d4809bb0b0.pdf"},{"id":90197568,"identity":"1f5f5227-5c47-4316-b8e9-c0d554e0d3a7","added_by":"auto","created_at":"2025-08-29 17:43:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29811,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable12025.06.13.docx","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/240a857d5531564174c1d5da.docx"},{"id":90197569,"identity":"ce38756f-d681-4432-a808-3bd98c8a473a","added_by":"auto","created_at":"2025-08-29 17:43:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":27505,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable22025.06.13.docx","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/60d6d1f04d97fb0eadb751ce.docx"},{"id":90197067,"identity":"693646b6-a8e3-4a4c-9cd4-95ff2b94f2d8","added_by":"auto","created_at":"2025-08-29 17:35:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":28312,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable32025.06.13.docx","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/395410125ea822be1f67f693.docx"},{"id":90197088,"identity":"ad918652-96b0-4a61-8c61-2cdaf58dda53","added_by":"auto","created_at":"2025-08-29 17:35:08","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4243466,"visible":true,"origin":"","legend":"","description":"","filename":"SourcedWBdata2025.08.03.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7187190/v1/bbf9fc6e10d00f87079a3b67.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"OTUD4 suppresses colorectal cancer progression through deubiquitinating p53","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most prevalent diagnosed malignancy and a leading cause of cancer-related mortality worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite advancements in surgical techniques and chemotherapeutic regimens, patient outcomes remain suboptimal due to high rates of metastasis, recurrence, and treatment resistance\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These challenges are compounded by the complex molecular landscape of CRC, which involves a multitude of genetic and proteomic alterations contributing to tumor initiation, progression, and relapse\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Key genetic mutations frequently observed in CRC include alterations in TP53, KRAS, and APC genes, as well as deficiencies in DNA mismatch repair mechanisms, leading to microsatellite instability\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, the precise molecular mechanisms driving CRC pathogenesis are not fully elucidated.\u003c/p\u003e\u003cp\u003eThe p53 transcription factor acts as an indispensable guardian against cancer, modulating a broad spectrum of cellular activities crucial for genomic integrity, including DNA damage repair, progression of cell cycle, induction of senescence, initiation of cell death pathways, facilitation of differentiation, and regulation of metabolic processes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. p53 and the related signaling is frequently dysregulated in colorectal cancer, leading to tumorigenesis and progression\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Other than missense mutation and transcription dysregulation, post-translational modifications, such as ubiquitination, phosphorylation, and acetylation, critically regulate the function of p53 in CRC\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Ubiquitination constitutes a pivotal post-translational modification event for p53 regulation. MDM2, an established ubiquitin ligase, promotes K48-linked polyubiquitination of p53, targeting it for subsequent degradation.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Besides, other E3 ligases such as COP1, UFL1, and FBXL6 also modulate p53 ubiquitination\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, the precise molecular mechanisms governing p53 ubiquitination which involving in CRC progression remain incompletely understood and warrant further elucidation.\u003c/p\u003e\u003cp\u003eOTU domain-containing protein 4 (OTUD4) is a deubiquitinating enzyme (DUB) belonging to the ovarian tumor (OTU) family, capable of hydrolyzing Lys48- and Lys63-linked polyubiquitin chains from modified proteins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. OTUD4 participates in diverse biological processes, including innate immune response\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, antiviral reaction\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, DNA damage and RNA binding\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In cancer, OTUD4 has been reported to promote glioblastoma\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, breast cancer\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, melanoma\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e progression via its deubiquitinating activity. Conversely, other studies indicated that OTUD4 acts as a tumor suppressor in intrahepatic cholangiocarcinoma\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, non-small cell lung cancer\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, ovarian tumor\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The context-dependent role of OTUD4 in tumor progression, whether oncogenic or tumor-suppressive, remains to be fully elucidated.\u003c/p\u003e\u003cp\u003eIn this study, we demonstrated the lower expression of OTUD4 in CRC tissues compared to adjacent normal tissues. OTUD4 suppresses CRC cells progression \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by deubiquitinating and stabilizing tumor suppressor p53. Collectively, our findings reveal a novel tumor-suppressive mechanism of OTUD4 by stabilizing p53, thereby inhibiting colorectal cancer development.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell lines and culture conditions\u003c/h2\u003e\u003cp\u003eThe human CRC cell line HCT116, murine CRC cell line CT26 and Human Embryonic Kidney 293T cells were purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). All the cell lines and were cultured at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e in in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Routine mycoplasma testing confirmed all lines were free of contamination.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePatients and tissue specimens\u003c/h3\u003e\n\u003cp\u003eTumor tissue microarrays of CRC patients were obtained from Department of Anorectal Surgery, the Second Hospital of Tianjin Medical University. All the samples and follow-up data acquisition were under the approval of Human Ethics Committee of Second Hospital of Tianjin Medical University (Approval Number: No.KY2023K207). Written informed consent was obtained from all the patients. All the methods were carried out in accordance with relevant guidelines.\u003c/p\u003e\n\u003ch3\u003eIHC Analysis\u003c/h3\u003e\n\u003cp\u003eAntibody detection was performed using standard IHC methods and the result of staining was evaluated using a semi-quantitative scoring system as previously described. The staining intensity (0: negative; 1: weak; 2: moderate; 3: strong) and percentage of positively stained cells (0: \u0026lt;5%; 1: 5–25%; 2: 26–50%; 3: 51–75%; 4: \u0026gt;75%) were determined independently. A composite IHC score (range 0–12) was calculated by multiplying the intensity score by the frequency score. The detailed information of antibodies is listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003ePlasmids and shRNA\u003c/h3\u003e\n\u003cp\u003eFull-length human \u003cem\u003eOTUD4\u003c/em\u003e cDNA (CCDS47139.1) and murine \u003cem\u003eOtud4\u003c/em\u003e (CCDS57625.1) was cloned into the vector with an N-terminal HA tag. Human \u003cem\u003eTP53\u003c/em\u003e cDNA (CCDS11118.1) was cloned into a pLenti vector with an N-terminal Flag tag. pLKO.1 vectors carrying shRNA targeting OTUD4 and p53 was purchased from Tsingke Biotechnology Company (Beijing, China). Indicated sequence of shRNA target are listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eLentiviral Production and Transduction\u003c/h3\u003e\n\u003cp\u003eLentiviral particles were produced in 293T cells via PEI-mediated transfection (40816ES02, Yeasen). Cells were transfected with 2 µg of lentiviral plasmid, 1.5 µg psPAX2, and 0.5 µg pMD2.0 G. Viral supernatant was harvested 48h and 72h post-transfection, pooled, filtered (0.45 µm), and used to infect CRC cells. Stable cell lines were selected using 1–5 µg/mL puromycin for at least two weeks.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation (IP)\u003c/h2\u003e\u003cp\u003eFor Co-IP, cells were washed with ice-cold PBS, harvested, and lysed using IP buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05 mM EDTA, 1% NP-40, 20% glycerol) supplemented with 1% protease/phosphatase inhibitor cocktail (Transgen, DI111-01). Lysate was clarified by centrifugation at 20,000g for 5 min at 4℃. Target proteins were then immunoprecipitated by incubating the primary bodies and pre-cleared protein-G magnetic beads at 4℃ overnight. After extensive washing with IP buffer, immunoprecipitated complexes were eluted for Western blotting or mass spectrometry (MS) analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eCells were lysed with denaturing buffer (1% SDS, 50 mM Tris-HCl Ph 7.4 and 2 mM EDTA in ddH2O) and then quantified by nanodrop after boiled for 10 min at 100℃. Proteins (40 µg per lane) were separated on SDS-PAGE, transferred to NC membranes (Millipore), blocked by 5% no-fat milk, and then incubated with primary antibodies overnight at 4℃. Following three washes, the membranes were incubated with HRP-conjugated secondary antibody and detected using enhanced chemiluminescence (ECL). Housekeep gene GAPDH as used as a loading control. The detailed information of antibodies is listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eubiquitination assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e ubiquitination assays were performed as previously described. In brief, 293T cells were co-transfected with His-Ub, HA-OTUD4 and Flag-p53 plasmids. cells were treated with 10 µM MG-132 (Sigma-Aldrich, 133407-82-6) for 4 h to inhibit proteasomal degradation. Cells were harvested and lysed in denaturing buffer (6 M guanidine-HCl, 100 mM Na₂HPO₄/NaH₂PO₄, 10 mM Tris-HCl, 5 mM imidazole, pH 8.0). Ubiquitinated proteins were enriched under denaturing conditions using Ni-NTA agarose beads (QIAGEN, 30210) to isolate His-ubiquitin conjugates. Ubiquitination of Flag-p53 was detected by immunoblotting with anti-Flag antibody.\u003c/p\u003e\u003cp\u003e\u003cb\u003eReverse transcription\u003c/b\u003e–\u003cb\u003equantitative PCR (RT\u003c/b\u003e–\u003cb\u003eqPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor RT–qPCR, total RNA was extracted using Trizol Reagent (Invitrogen) and was reverse transcripted to cDNA by HiScript II Reverse Transcriptase kit (Vazyme, R211). SYBR Green Supermix (Vazyme, Q712) was used to detect the corresponding mRNA level using and GAPDH was used as an internal control. All the sequence of primers used in this article were listed in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eCell cycle and apoptosis analysis\u003c/h3\u003e\n\u003cp\u003eFor cell cycle analysis, cells were collected after twice washing by cold PBS and fixed with 70% ethanol overnight at 4℃. Then cells were resuspended with RNase A and propidium iodide (PI, Beyotime, Y267501) for 20 min at room temperature and then analyzed by detecting PerCP 5.5 using flow cytometry. For apoptosis analysis, cells were collected with trypsin without EDTA and then stained with Annexin V and PI. The cells were analyzed by detecting FITC and PerCP 5.5 using flow cytometry.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCCK-8 assays, Colony formation and Transwell assays\u003c/h2\u003e\u003cp\u003eFor CCK-8 assays, 1000 cells were seeded into 96-well plates with 100 µl complete DMEM medium. The cell numbers were counted by CCK-8 reagent (Beyotime, C0037) using a microplate spectrophotometer at different points in time. Absorbance at 450 nm was employed to determine the number of viable cells and cell viability was calculated based on a standard curve. For colony formation assay, 300 cells were seeded in 6-well plates with 3 mL complete DMEM medium. After 10–14 days, colonies were washed with PBS, fixed with 100% methanol for 15 min, and stained with 0.5% crystal violet for 30 min at room temperature. Crystal violet (Beyotime, Y268091) stained colonies (\u0026gt; 50 cells/colony) were automatically quantified using ImageJ v1.53. For Transwell assays, Cell migration was assessed using 8-µm pore Transwell inserts (Corning). 5 × 10⁴ cells in 200 µL serum-free DMEM were seeded into the upper chamber. The lower chamber contained 500 µL DMEM with 20% FBS. After 24 h incubation, migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted in 5 random fields per insert (100 × magnification).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnimal study\u003c/h2\u003e\u003cp\u003e Tumor xenograft studies were approved by the Institutional Animal Care and Use Committee, and animal experiments were performed in accordance with protocols approved by the Second Hospital of Tianjin Medical University Animal Care and Use Committee. (No. TMU2024119). The 6-week-old male nude mice were purchased from Cyagen Biosciences (Guangzhou, China). For experiments, 5 mice per group would be sufficient to detect a difference based on previous experience with the model, and animals were randomly assigned to different groups. Investigators were not blinded to group allocation. SPF mice were maintained under specific pathogen-free conditions with controlled temperature (22–24°C), humidity (40–70%), sterile feed/water, and IVC housing, following institutional animal care protocols. CT26 cells expressing vector control or OTUD4 (1×10\u003csup\u003e6\u003c/sup\u003e cells in 100 µL PBS) were injected subcutaneously into the right flank of mice after being anesthetized via intraperitoneal injection of tert-amyl alcohol/tribromoethanol. Tumor volume was measured every 3 days using digital calipers from day 7 post-injection until day 16, calculated as: Tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) = 0.5 × Length × Width\u003csup\u003e2\u003c/sup\u003e. On day 16, mice were euthanized by CO₂ asphyxiation followed by cervical dislocation, and tumors were excised. The maximal tumor size burden (20% in any dimension or 10% of body weight) was not exceeded, which was permitted by the ethics committee. All the tumor size in animals is less than 500 mm³, which generally does not cause pain, weight loss, or physiological distress in mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e\u003cp\u003eAll the \u003cem\u003eex vivo\u003c/em\u003e experiments were repeated at least three times and data are presented as the mean ± SD. The data presentation and statistical analyses are described in the figure legends. Differences with \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism 9.0 Software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com/features\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com/features\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe cDNAs used during the current study are available in the PubMed repository (human \u003cem\u003eOTUD4\u003c/em\u003e: CCDS47139.1; Murine \u003cem\u003eOtud4\u003c/em\u003e: CCDS57625.1; Human \u003cem\u003eTP53\u003c/em\u003e: CCDS11118.1.), [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/gene\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/gene\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003eLower OTUD4 Expression Correlates with Poor Prognosis in Colorectal Cancer\u003c/h2\u003e\u003cp\u003eTo investigate the dysregulation of OTUD4 in colorectal cancer (CRC), we first assessed its mRNA expression using the GEPIA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and found that both colon and rectal carcinoma tissues exhibited significantly lower OTUD4 mRNA levels compared to matched normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Confirming these findings at the protein level, proteomic analysis of the International Cancer Proteogenome Consortium (ICPC) datasets revealed a marked reduction of OTUD4 in tumor tissues relative to adjacent normal counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, IHC staining of tissue microarrays (TMAs) from our clinical cohort revealed notably diminished OTUD4 protein expression in CRC samples compared to that in normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). We next examined the prognostic implications of reduced OTUD4 expression. We found that CRC patients with lower OTUD4 expression had a shorter relapse-free survival (RFS) and overall survival (OS) using the TCGA dataset via the Kaplan–Meier Plotter platform [CRC] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kmplot.com/analysis\u003c/span\u003e\u003cspan address=\"https://kmplot.com/analysis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and GEPIA data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/detail.php?gene=OTUD4\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/detail.php?gene=OTUD4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). Together, these results suggest that OTUD4 is downregulated in CRC and is associated with poorer prognosis.\u003c/p\u003e\u003ch2\u003eOTUD4 Suppresses CRC Cell Proliferation, Promotes Apoptosis, and Inhibits Migration\u003c/h2\u003e\u003cp\u003eTo investigate the function of OTUD4 in CRC cells, the OTUD4 knockdown and control HCT116 cells were constructed using lentiviral. The cell cycle assay results revealed that OTUD4 knockdown significantly decreased the proportion of cells in G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Correspondingly, Annexin V assays demonstrated a marked decrease in apoptotic cell population upon OTUD4 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, CCK8 and clonal formation assays demonstrated that OTUD4 negatively regulated the growth of HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Additionally, Transwell assay showed that knockdown of OTUD4 enhanced the migration ability of HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these data indicate that OTUD4 acts as a tumor suppressor by restraining cell cycle progression, enhancing apoptosis, and inhibiting proliferation and migration in CRC cells.\u003c/p\u003e\u003ch2\u003eOTUD4 Deubiquitinates and Stabilizes p53 to in CRC\u003c/h2\u003e\u003cp\u003eTo elucidate the mechanism by which OTUD4 suppresses CRC, we overexpressed HAtagged OTUD4 in HCT116 cells, performed immunoprecipitation, and identified interacting substrates via mass spectrometry. Notably, the tumor suppressor p53 emerged among OTUD4-binding proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Accordingly, we hypothesized that OTUD4 stabilizes p53 through its deubiquitinase activity.\u003c/p\u003e\u003cp\u003eEndogenous OTUD4–p53 interaction was confirmed in wildtype p53 HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Subsequent in vivo ubiquitylation assays revealed that OTUD4 overexpression markedly diminished polyubiquitylation of p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To determine whether OTUD4 influences p53 protein stability, we overexpressed OTUD4 in CT26 cells, observing increased p53 levels, while OTUD4 knockdown reduced p53 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistently, OTUD4 overexpression substantially extended p53 half-life in CT26 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Further, OTUD4 depletion in HCT116 cells led to decreased levels of cleaved caspase‑3, while OTUD4 overexpression enhanced caspase‑3 cleavage. Correspondingly, both mRNA and protein levels of the canonical p53 target gene p21 increased following OTUD4 overexpression—despite p53 mRNA remaining unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF–I). Crucially, shRNA-mediated p53 knockdown abolished OTUD4-induced upregulation of cleaved caspase‑3 and p21, thereby establishing the dependence on p53. Collectively, these data show that OTUD4 directly deubiquitinates p53, enhancing its protein stability and triggering p53-dependent transcriptional activation of apoptosis and cell-cycle arrest pathways.\u003c/p\u003e\u003ch2\u003eOTUD4 Suppresses CRC Cell Aggressiveness in a p53-Dependent Manner\u003c/h2\u003e\u003cp\u003eNext, we constructed OTUD4-overexpressing, OTUD4-overexpressing with p53-knockdown and corresponding control CT26 cells. As expected, overexpression of OTUD4 decreased the portion of cells in G2/M phase and decreased the portion of annexin V-positive apoptotic cells, while knockdown of p53 reversed the effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). Functionally, overexpression of OTUD4 inhibited CT26 cell proliferation and migration; these suppressive effects were again abrogated by p53 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC–E). Taken together, our data demonstrate that OTUD4 attenuates the aggressiveness of colorectal cancer cells in a p53-dependent manner—reinforcing the critical role of the OTUD4–p53 axis in CRC suppression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOverexpression of OTUD4 inhibited the growth of xenograft tumors in mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the \u003cem\u003ein vivo\u003c/em\u003e effects of OTUD4 on colorectal cancer, we established subcutaneous xenografts in NOD-SCID mice using CT26 cells stably overexpress OTUD4 or empty vector control. Tumor volumes were measured every three days using calipers. Tumors derived from OTUD4\u003csup\u003eOE\u003c/sup\u003e cells exhibited significantly reduced growth compared to control tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA–C). For the further, IHC assays on xenografts tumors revealed elevated p53 protein levels in OTUD4\u003csup\u003eOE\u003c/sup\u003e tumors relative to vector controls. Concurrently, proliferation marker Ki‑67 was diminished, and apoptosis marker Cleaved Caspase-3 was markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results confirm that OTUD4 overexpression suppresses colorectal tumor growth in vivo, likely through stabilization of p53 leading to reduced proliferation and increased apoptosis, consistent with its function as a tumor suppressor in CRC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we comprehensively investigated OTUD4 in CRC and elucidated its crucial role as a tumor suppressor. Our findings reveal that OTUD4 is downregulated at both mRNA and protein levels in CRC tissues compared to normal colon and rectal tissues, with lower expression correlated with significantly worse relapse-free and overall survival. Functional assays demonstrated OTUD4\u0026rsquo;s suppression of proliferation, cell cycle progression, migration, and its promotion of apoptosis in vitro, as well as its inhibition of tumor growth in vivo. Mechanistically, OTUD4 directly binds p53, reduces its ubiquitination, stabilizes p53, and enhances p53-dependent transcription leading to cell-cycle arrest and apoptosis. Importantly, OTUD4\u0026rsquo;s anti-tumor functions are p53-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eP53 is one of the most important tumor suppressors in CRC. The CDKN1A gene, coding for the cyclin-dependent kinase inhibitor p21, was the first discovered transcriptional target of p53. Other than the suppression function of transformation and proliferation by direct inhibiting CDKs, p53-p21 regulatory axis also promotes metastasis of CRC cells via EMT promoting\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. On the other side, p53 promotes CRC cells apoptosis by activating Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP activation and Bcl2 deactivation, thereby inhibiting tumor proliferation and promoting apoptosis\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We found that OTUD4 promotes the activation of cleaved caspase-3 as well as the transcription of CDKN1A (p21) by increasing the p53 protein level. These results position OTUD4 as a novel DUB that reinforces tumor suppressor pathways via p53 stabilization in CRC. This mechanism mirrors other OTU-family DUBs, such as OTUD1\u003csup\u003e32\u003c/sup\u003e, OTUD3\u003csup\u003e33\u003c/sup\u003e and OTUD5\u003csup\u003e34\u003c/sup\u003e, modulating of p53 activity, and is consistent with previous evidence that many DUBs regulate cell proliferation, cycle control, and apoptosis in cancer progression\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our work corroborates broader findings that deubiquitination plays a vital role in maintaining protein homeostasis in CRC.\u003c/p\u003e\u003cp\u003eNotably, OTUD4\u0026rsquo;s function in CRC appears to diverge from its role in other cancers. For instance, deubiquitylation of CD73 by OTUD4 results in CD73 stabilization and inhibit tumor immune responses of triple negative breast cancer\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In various type of tumors, OTUD4 not only sustains protein stability by directly deubiquitinating GPX4 but also impedes its degradation via RHEB-mediated autophagy, collectively stalling the ferroptosis pathway\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These findings highlight the context-dependent specificity of OTUD4 substrates across tissues. In CRC, OTUD4 appears to function primarily through the p53 axis, reinforcing p53\u0026rsquo;s stability and activity\u0026mdash;a central tumor suppressive pathway commonly inactivated in CRC.\u003c/p\u003e\u003cp\u003eMoreover, our \u003cem\u003ein vivo\u003c/em\u003e data further support OTUD4\u0026rsquo;s biological relevance. In CT26 xenografts, OTUD4 overexpression decreased tumor growth and proliferation (Ki-67 expression), while increasing p53 levels and apoptosis (cleaved caspase-3). These \u003cem\u003ein vivo\u003c/em\u003e phenotypes underscore OTUD4's potential clinical importance.\u003c/p\u003e\u003cp\u003eFrom a translational perspective, our findings indicate that OTUD4 expression or activity could serve as a prognostic biomarker or therapeutic entry point. Given that DUB-targeted drugs have been explored in oncology pharmacological activation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e or gene delivery of OTUD4 might represent a strategy to reactivate p53 signaling in CRC with intact p53. Conversely, in tumors where OTUD4 supports oncogenic pathways, targeted inhibition may be beneficial. Thus, elucidating OTUD4\u0026rsquo;s substrate specificity and regulation in CRC could guide tailored therapeutic applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eEthics statement\u003c/h2\u003e\n\u003cp\u003eTumor tissue microarrays of CRC patients were obtained from Department of Anorectal Surgery, the Second Hospital of Tianjin Medical University. All the samples and follow-up data acquisition were under the approval of Human Ethics Committee of Second Hospital of Tianjin Medical University (Approval Number: No.KY2023K207). Written informed consent was obtained from all the patients. All methods were carried out in accordance with relevant guidelines.\u003c/p\u003e\n\u003cp\u003eTumor xenograft studies were approved by the Institutional Animal Care and Use Committee, and animal experiments were performed in accordance with protocols approved by the Second Hospital of Tianjin Medical University Animal Care and Use Committee. (No. TMU2024119). The maximal tumor size burden (20% in any dimension or 10% of body weight) was not exceeded, which was permitted by the ethics committee. All the tumor size in animals is less than 500 mm\u0026sup3;, which generally does not cause pain, weight loss, or physiological distress in mice.\u003c/p\u003e\n\u003ch2\u003eConsent to publish\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors confirm that this investigation was executed without any commercial or financial associations that might represent a potential conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo funding to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eXYB, YXW, JZP and BZ wrote the main manuscript text ; XYB, YXW and JZP prepared figures 1-5, SL and PHL prepared figure 6. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaidoun F, et al. Colorectal Cancer Epidemiology: Recent Trends and Impact on Outcomes. Curr Drug Targets. 2021;22:998\u0026ndash;1009. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2174/1389450121999201117115717\u003c/span\u003e\u003cspan address=\"10.2174/1389450121999201117115717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEng C, et al. A comprehensive framework for early-onset colorectal cancer research. 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Biomed Pharmacother. 2024;175:116753. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.biopha.2024.116753\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2024.116753\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CRC, OTUD4, p53, Cancer progression, Deubiquitination","lastPublishedDoi":"10.21203/rs.3.rs-7187190/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7187190/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) represents a global health burden as the third most commonly diagnosed malignancy and the second leading cause of cancer-related mortality. Despite advances in research, molecular mechanisms driving CRC initiation, progression, and metastasis remain incompletely elucidated. OTU domain-containing protein 4 (OTUD4), a deubiquitinating enzyme of the ovarian tumor family, catalyzes ubiquitin chain removal from substrate proteins. While implicated in diverse cellular processes, OTUD4's role in CRC pathogenesis is undefined. Here, we report significant downregulation of OTUD4 in CRC specimens relative to normal colonic epithelium, and demonstrate OTUD4 depletion enhances proliferation, clonogenicity, migration, and invasion in CRC cell lines, consistent with tumor-suppressive activity. Mechanistically, OTUD4 interacts with and deubiquitinates p53, thereby stabilizing this tumor suppressor protein and enhancing its transcriptional activity. Critically, p53 knockdown abrogates OTUD4-mediated suppression of malignant phenotypes, establishing the OTUD4-p53 axis as a critical regulatory node in CRC. Our findings identify OTUD4 as a novel tumor suppressor that constrains colorectal carcinogenesis through deubiquitinating and stabilizing p53, highlighting its therapeutic potential and warranting deeper investigation of OTUD4 in cancer biology.\u003c/p\u003e","manuscriptTitle":"OTUD4 suppresses colorectal cancer progression through deubiquitinating p53","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 17:35:02","doi":"10.21203/rs.3.rs-7187190/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-29T10:28:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T16:21:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8433241857833131976694018631401622802","date":"2025-09-18T11:36:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-08T13:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125439472948793088179554690785948498354","date":"2025-08-28T08:31:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-21T12:54:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-21T12:52:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-14T18:06:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-08T09:22:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2025-08-08T09:19:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4318d36-8d71-4213-93d9-071a86734b14","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-05T09:53:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 17:35:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7187190","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7187190","identity":"rs-7187190","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}