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The deubiquitinase USP25 supports colonic inflammation and bacterial infection and promotes colorectal cancer

Nature Cancer volume 1, pages 811–825 (2020)Cite this article

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Abstract

Bacterial infection or abnormal colonization in the gastrointestinal system is associated with subsets of inflammatory bowel disease and colorectal cancer. Here we demonstrated essential roles of ubiquitin-specific protease 25 (USP25) in experimental colitis, bacterial infections and colon cancer. Knockout or pharmacologic inhibition of USP25 potentiated immune responses after induction of experimental colitis or bacterial infections that promoted clearance of infected bacteria and resolution of inflammation and attenuated Wnt and SOCS3–pSTAT3 signaling, which inhibited colonic tumorigenesis. USP25 levels were positively or negatively correlated with Fusobacterium nucleatum colonization and β-catenin levels or SOCS3 levels in human colorectal tumor biopsies, respectively, and predicted poor prognosis of patients with cancers in the gastrointestinal system. Our findings suggest USP25 as a promoter and druggable target for gastrointestinal infections and cancers.

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Fig. 1: USP25 deficiency leads to resistance to DSS-induced colitis.
Fig. 2: USP25 deficiency leads to altered cytokine expression.
Fig. 3: Usp25−/− mice exhibit resistance to bacterial infections.
Fig. 4: USP25 deficiency inhibits tumorigenesis in the colon.
Fig. 5: USP25 deficiency impairs Wnt signaling and upregulates Socs3.
Fig. 6: Targeting USP25 impairs DSS-induced colitis and restricts bacterial infections.
Fig. 7: USP25 is a therapeutic target for colon cancer.

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Data availability

The RNA-seq and 16S rRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE136727 and GSE143003, which can be found in Supplementary Tables 1 and 4, respectively.

Hhuman GI cancer data were derived from GEPIA (gepia.cancer-pku.cn). The dataset derived from this resource that supports the findings of this study is available at http://gepia.cancer-pku.cn/detail.php?gene=USP25### (cutoff-high, 75%, cutoff-low, 25%; datasets: COAD, READ, STAD). Source data for Figs. 1–7 and Extended Data Fig. 1–8 have been provided as Source Data Figs. 1–7 (numerical source data), Source Data Extended Data Figs. 1–8 (numerical source data), Source Data Figs. 1–3 and 6–7 (uncropped gels) and Source Data Extended Data Figs. 2 and 5 (uncropped gels). All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Kaser, A., Zeissig, S. & Blumberg, R. S. Inflammatory bowel disease. Annu. Rev. Immunol. 28, 573–621 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Somineni, H. K. & Kugathasan, S. The microbiome in patients with inflammatory diseases. Clin. Gastroenterol. Hepatol. 17, 243–255 (2019).

    Article  PubMed  Google Scholar 

  3. Jostins, L. et al. Host–microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 491, 119–124 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hugot, J. P. et al. Association of Nod2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 411, 599–603 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Ogura, Y. et al. A frameshift mutation in Nod2 associated with susceptibility to Crohn’s disease. Nature 411, 603–606 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Kobayashi, K. S. et al. Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science 307, 731–734 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Maeda, S. et al. Nod2 mutation in Crohn’s disease potentiates NF-κB activity and IL-1β processing. Science 307, 734–738 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Sokol, H. et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc. Natl Acad. Sci. USA 105, 16731–16736 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Darfeuille-Michaud, A. et al. Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease. Gastroenterology 115, 1405–1413 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Ohkusa, T. et al. Induction of experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa of patients with ulcerative colitis. Gut 52, 79–83 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J. Clin. 68, 394–424 (2018).

    Google Scholar 

  12. Lasry, A., Zinger, A. & Ben-Neriah, Y. Inflammatory networks underlying colorectal cancer. Nat. Immunol. 17, 230–240 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Dow, L. E. et al. APC restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer. Cell 161, 1539–1552 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Greten, F. R. et al. IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118, 285–296 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Schwitalla, S. et al. Loss of p53 in enterocytes generates an inflammatory microenvironment enabling invasion and lymph node metastasis of carcinogen-induced colorectal tumors. Cancer Cell 23, 93–106 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Tauriello, D. V. F. et al. TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis. Nature 554, 538–543 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. Cell 124, 783–801 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Walsh, D., McCarthy, J., O’Driscoll, C. & Melgar, S. Pattern-recognition receptors–molecular orchestrators of inflammation in inflammatory bowel disease. Cytokine Growth Factor Rev. 24, 91–104 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by Toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Fukata, M. et al. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G1055–G1065 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Dheer, R. et al. Intestinal epithelial Toll-like receptor 4 signaling affects epithelial function and colonic microbiota and promotes a risk for transmissible colitis. Infect. Immun. 84, 798–810 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhong, B. et al. Ubiquitin-specific protease 25 regulates TLR4-dependent innate immune responses through deubiquitination of the adaptor protein TRAF3. Sci. Signal. 6, ra35 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Lin, D. et al. Induction of USP25 by viral infection promotes innate antiviral responses by mediating the stabilization of TRAF3 and TRAF6. Proc. Natl Acad. Sci. USA 112, 11324–11329 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xu, D. et al. USP25 regulates Wnt signaling by controlling the stability of tankyrases. Genes Dev. 31, 1024–1035 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kinchen, J. et al. Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease. Cell 175, 372–386 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tabula Muris, C. et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 562, 367–372 (2018).

    Article  CAS  Google Scholar 

  27. Kitajima, S., Takuma, S. & Morimoto, M. Changes in colonic mucosal permeability in mouse colitis induced with dextran sulfate sodium. Exp. Anim. 48, 137–143 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Yang, Y. et al. Fusobacterium nucleatum increases proliferation of colorectal cancer cells and tumor development in mice by activating Toll-like receptor 4 signaling to nuclear factor-κB, and up-regulating expression of microRNA-21. Gastroenterology 152, 851–866 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. Abed, J. et al. Fap2 mediates Fusobacterium nucleatum colorectal adenocarcinoma enrichment by binding to tumor-expressed Gal-GalNAc. Cell Host Microbe 20, 215–225 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Rubinstein, M. R. et al. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin. Cell Host Microbe 14, 195–206 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kostic, A. D. et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 14, 207–215 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yu, T. et al. Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. Cell 170, 548–563 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tomkovich, S. et al. Locoregional effects of microbiota in a preclinical model of colon carcinogenesis. Cancer Res. 77, 2620–2632 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Suzuki, A. et al. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation. J. Exp. Med. 193, 471–481. (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, Y. et al. Disease-related expression of the IL6/STAT3/SOCS3 signalling pathway in ulcerative colitis and ulcerative colitis-related carcinogenesis. Gut 59, 227–235 (2010).

    Article  PubMed  CAS  Google Scholar 

  36. Wrigley, J. D. et al. Identification and characterization of dual inhibitors of the USP25/28 deubiquitinating enzyme subfamily. ACS Chem. Biol. 12, 3113–3125 (2017).

    Article  CAS  PubMed  Google Scholar 

  37. Parikh, K. et al. Colonic epithelial cell diversity in health and inflammatory bowel disease. Nature 567, 49–55 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Schmitt, M. et al. Paneth cells respond to inflammation and contribute to tissue regeneration by acquiring stem-like features through SCF/c-Kit signaling. Cell Rep. 24, 2312–2328 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Adolph, T. E., Mayr, L., Grabherr, F. & Tilg, H. Paneth cells and their antimicrobials in intestinal immunity. Curr. Pharm. Des. 24, 1121–1129 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Price, A. E. et al. A map of Toll-like receptor expression in the intestinal epithelium reveals distinct spatial, cell type-specific, and temporal patterns. Immunity 49, 560–575 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bersudsky, M. et al. Non-redundant properties of IL-1α and IL-1β during acute colon inflammation in mice. Gut 63, 598–609 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. Hasegawa, M. et al. Protective role of commensals against Clostridium difficile infection via an IL-1β-mediated positive-feedback loop. J. Immunol. 189, 3085–3091 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Gao, S. J. et al. Interleukin-18 genetic polymorphisms contribute differentially to the susceptibility to Crohn’s disease. World J. Gastroentero. 21, 8711–8722 (2015).

    Article  CAS  Google Scholar 

  44. Harrison, O. J. et al. Epithelial-derived IL-18 regulates Th17 cell differentiation and Foxp3(+) Treg cell function in the intestine. Mucosal Immunol. 8, 1226–1236 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Tak, T., Tesselaar, K., Pillay, J., Borghans, J. A. & Koenderman, L. What’s your age again? Determination of human neutrophil half-lives revisited. J. Leukoc. Biol. 94, 595–601 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Sylvia, C. J. The role of neutrophil apoptosis in influencing tissue repair. J. Wound Care 12, 13–16 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Hart, J. Inflammation. 1: its role in the healing of acute wounds. J. Wound Care 11, 205–209 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Zhao, J., Kim, K. A. & Abo, A. Tipping the balance: modulating the Wnt pathway for tissue repair. Trends Biotechnol. 27, 131–136 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Bradford, E. M. et al. Epithelial TNF receptor signaling promotes mucosal repair in inflammatory bowel disease. J. Immunol. 199, 1886–1897 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Isomoto, H. et al. Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology 132, 384–396 (2007).

    Article  CAS  PubMed  Google Scholar 

  51. Carow, B. & Rottenberg, M. E. SOCS3, a major regulator of infection and inflammation. Front. Immunol. 5, 58 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Brennan, C. A. & Garrett, W. S. Gut microbiota, inflammation, and colorectal cancer. Annu. Rev. Microbiol. 70, 395–411 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhong, B. et al. Negative regulation of IL-17-mediated signaling and inflammation by the ubiquitin-specific protease USP25. Nat. Immunol. 13, 1110–1117 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhao, Y. et al. USP2a supports metastasis by tuning TGF-β signaling. Cell Rep. 22, 2442–2454 (2018).

    Article  CAS  PubMed  Google Scholar 

  55. Yang, X. O. et al. Regulation of inflammatory responses by IL-17F. J. Exp. Med. 205, 1063–1075 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Li, S. et al. Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains. Nature 501, 242–246 (2013).

    Article  CAS  PubMed  Google Scholar 

  57. Zhang, M. et al. USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA. Cell Res. 26, 1302–1319 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Liuyu, T. et al. Induction of OTUD4 by viral infection promotes antiviral responses through deubiquitinating and stabilizing MAVS. Cell Res. 29, 67–79 (2019).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Y. Pan (Fourth Military Medical University) for valuable suggestions, S. Li (Huazhong Agricultural University) for reagents and technical help and members of the Zhong laboratory and the core facilities of the Medical Research Institute for technical help. This study was supported by grants from the National Key Research and Development Program of China (2018TFE0204500 and 2018YFC1004601), Natural Science Foundation of China (31671454 and 31930040), Fundamental Research Funds for Central Universities (2042020kf0207 and 2042020kf0042), Natural Science Foundation of Hubei Province (2018CFA016) and Medical Science Advancement Program (Basic Medical Sciences) of Wuhan University (TFJC2018004).

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Authors and Affiliations

  1. Department of Gastrointestinal Surgery, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan, China

    Xiao-Meng Wang, Ci Yang, Yin Zhao, Wei Yang, Peng Wang, Bin Xiong & Bo Zhong

  2. Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China

    Xiao-Meng Wang, Ci Yang, Yin Zhao, Wei Yang, Peng Wang & Bo Zhong

  3. Department of Virology, College of Life Sciences, Wuhan University, Wuhan, China

    Xiao-Meng Wang, Ci Yang, Wei Yang, Peng Wang & Bo Zhong

  4. Institute of Hepatobiliary Diseases, Transplant Center, Hubei Key Laboratory of Medical Technology on Transplantation, Zhongnan Hospital of Wuhan University, Wuhan, China

    Zhi-Gao Xu

  5. Cancer Center, Renmin Hospital of Wuhan University, Wuhan, China

    Dandan Lin

  6. Division of Gastroenterology and Hepatology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Jing-Yuan Fang

  7. Institute for Immunology and School of Medicine, Tsinghua University, Beijing, China

    Chen Dong

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Contributions

B.Z. designed and supervised the study; X.-M.W. performed the core experiments; C.Y. and Y.Z. helped with mouse breeding and colon cancer modeling; Z.X. and P.W. performed bioinformatics analysis; Z.-G.X., W.Y., D.L. and B.X. collected CRC samples, produced tissue arrays and performed IHC analysis; J.-Y.F. and C.D. provided reagents; B.Z., X.-M.W. and D.L. wrote the paper; all the authors analyzed data.

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Correspondence to Bo Zhong.

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Extended data

Extended Data Fig. 1 USP25 deficiency inhibits DSS-induced colitis independently of type I IFN signaling.

(a, b) Body weight change (a) and gross morphological change and lengths of colons (b) of Usp25-/-Ifnar1-/- mice (n = 3) and Ifnar1-/- mice (n = 3) that were given 2% DSS in drinking water for 5 d, followed by normal drinking water for another 4 d. (c) Representative histological images (left) and pathological scores (right) of H&E stained colon sections of Usp25-/-Ifnar1-/- mice (n = 3) and Ifnar1-/- mice (n = 3) in (a). (d–f) Total cell number (d), representative flow cytometry images and analysis of the percentages and cell number of neutrophils (CD11b+Gr-1+) and macrophages (CD11b+F4/80+) (E), CD8+, CD4+, CD3+CD4+IFNγ+, CD3+CD4+IL−17γ+, and CD3+CD4+Foxp3+ cells (f) from lamina propria in Usp25+/+ (n = 3) and Usp25-/- (n = 3) mice that were given 2.5% DSS in drinking water for 5 d, followed by normal drinking water for another 3 d. (g–i) Total cell number (g), representative flow cytometry images and analysis of the percentages and cell number of neutrophil (CD11b+Gr-1+) and macrophage (CD11b+F4/80+) (h), CD8+, CD4+, CD3+CD4+IFNγ+, CD3+CD4+IL-17γ+, and CD3+CD4+Foxp3+ cells (i) of lamina propria from Usp25-/-Ifnar1-/- mice (n = 3) and Ifnar1-/- mice (n = 3) that were given 2% DSS in drinking water for 5 d, followed by normal drinking water for another 3 d. *P < 0.05; **P < 0.01 (two-tailed student’s t-test). Scale bars represent 0.4 mm. Data are representative of two independent experiments (Graphs show mean ± S.D.). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.1.

Source data

Extended Data Fig. 2 USP25 deficiency in hematopoietic cells does not affect immune cell homeostasis or differentiation in the colon lamina propria after DSS treatment.

(a) Flow cytometry analysis of the blood from irradiated CD45.1+ mice that were adoptively transferred Usp25+/+ (n = 5) and Usp25-/- (n = 5) bone marrow cells (1×106) (CD45.2+) for 6 weeks. (b–d) Total cell number (b), representative flow cytometry images and analysis of the percentages and cell number of neutrophil (CD11b+Gr-1+) and macrophage (CD11b+F4/80+) (c), CD8+, CD4+, CD3+CD4+IFNγ+, CD3+CD4+IL-17γ+, and CD3+CD4+Foxp3+ cells (d) of lamina propria from mice in (a) that were given 2.5% DSS in drinking water for 5 d, followed by normal drinking water for another 3 d. (e) qRT-PCR analysis of Usp25 mRNA levels (left graph) (n = 4 for each time point) and immunoblot analysis (n = 2 for each time point) of USP25 protein in colon epithelial cells isolated from wild-type mice treated with 2.5% DSS for 0–4 d. Data are representative of two independent experiments (Graphs show mean ± S.D., two-tailed student’s t-test.). Uncropped immunoblot images are shown in Uncropped_gels_Extended_Data_Fig.2. Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.2.

Source data

Extended Data Fig. 3 USP25 deficiency inhibits DSS-induced colitis dependently on gut bacteria.

(a) Gene set enrichment analysis of IRF-dependent genes of Usp25+/+ (n = 2) and Usp25-/- (n = 2) mice that were given 2.5% DSS in drinking water for 5 d, followed by normal drinking water for another 2 d. (b) qRT-PCR analysis of IRF-dependent genes in the colons of Usp25+/+ (n = 2 at day 0, n = 4 at day 4 and 6) and Usp25-/- (n = 2 at day 0, n = 4 at day 4 and 6) mice that were given 2.5% DSS in drinking water for 4 or 6 d. (c) qRT-PCR analysis of the indicated genes in the colons from Usp25-/-Ifnar1-/- (n = 3) and Ifnar1-/- (n = 3) mice that were given 2% DSS in drinking water for 5 d, followed by normal drinking water for another 3 d. (d) A scheme of antibiotics treatment and colitis induction. Mice were fed with sterile water containing ampicillin (1 g/L), metronidazole (1 g/L), vancomycin (0.5 g/L), and neomycin (1 g/L). Four weeks later, the mice were used for 2% DSS-colitis analysis. (e) Survival of Usp25+/+ (n = 6) and Usp25-/- (n = 8) mice treated as in (d). (f, g) Body weight change (f), gross morphological change and lengths of colons, and representative H&E stained colon sections (g) of Usp25+/+ (n = 7) and Usp25-/- (n = 6) mice that were treated as in (d). (h) qRT-PCR analysis of the indicated genes in the colons of Usp25+/+ (n = 3) and Usp25-/- (n = 3) mice that were treated as in (d). *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed student’s t-test). Scale bars represent 0.4 mm. Data are representative of two (b, c, h) and combined two (e–g) independent experiments (Graphs show mean ± S.D.). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.3.

Source data

Extended Data Fig. 4 USP25 deficiency potentiates bacterial infection-induced expression of cytokines.

(a) Image (left) and cell number (right) of spleens from Usp25+/+ (n = 4) and Usp25-/- (n = 4) mice that were orally infected with C rodentium (1 ×109 CFU) for 14 days. (b, c) Flow cytometry analysis of spleenocytes strained with fluorescence-conjugated antibodies against the indicated surface from mice in (a) (n = 4). (d, e) qRT-PCR analysis of the expression of the indicated genes in the colons from Usp25+/+ (n = 3) and Usp25-/- (n = 3) mice that were orally infected with C rodentium (1 ×109 CFU) for 14 days (d) (n = 3 for Usp25+/+; n = 4 for Usp25-/-) or S typhimurium (1 ×107 CFU) for 6 days (e) (n = 5). (f, g) qRT-PCR analysis of the expression of the indicated genes in Usp25+/+ and Usp25-/- colon organoids (n = 3, technical replicates) that were infected with C rodentium and S typhimurium (CFU of 5×106 or 1×107) for 1 h (f) or stimulated with LPS (10 mg/ml) for 0–8 h (g). (h) Immunohistochemistry analysis (IHC) of USP25 and fluorescent in situ hybridization (FISH) of F. nucleatum in human colorectal cancer tissues (n = 18 for the + or - groups). (i) Correlation analysis (left) and representative images (right) of USP25 protein levels (IHC) and F. nucleatum colonization (FISH) in CRC tissues (n = 18). Scale bars represent 0.2 mm. (j) qRT-PCR analysis of the expression of USP25 in the colons from Usp25+/+mice day (n = 4 for each time point) that were orally infected with C rodentium (1 ×109 CFU) at 0, 5, 10 day. (k) qRT-PCR analysis of the expression of USP25 in the colons from Usp25+/+mice day (n = 4 for each time point) that were orally infected S typhimurium (1 ×107 CFU) at 0, 2, 4. (l) Representative images and tumor counts of colons from ApcMin/+ (n = 10) and ApcMin/+ Usp25-/- (n = 7) mice that were daily fed with F. nucletum (1 ×108 CFU) for 8 weeks beginning at 6 weeks of age. (m, n) FISH (m) and statistical (n) (n = 10 mice for ApcMin/+; n = 3 mice for ApcMin/+ Usp25-/-) analysis of F. nucleatum of colon tumors from mice in (l). Black scale bars represent 2 mm. Red scale bars represent 0.1 mm. *P < 0.05; **P < 0.01; ***P < 0.001 (two-way ANOVA for j and k; two-tailed student’s t-test for a–e, h, l and n). Data are representative of two independent experiments (Graphs show mean in f and g or mean ± S.D. in a–e, h, j–n). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.4.

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Extended Data Fig. 5 Negative correlation of USP25 expression level and prognosis of gastrointestinal cancers.

(a) The correlation of USP25 transcript expression with overall survival in gastrointestinal cancer patients (n = 187 for USP25 high and low patients, GEPIA database). (b) Immunoblot analysis of USP25 in the colon tumors from Usp25+/+ mice after AOM/DSS induction for 12 weeks (left), Vil-Cre;Trp53fl/fl mice at the 20th week after the first AOM injection (middle), or ApcMin/+ mice at 20-week-old (right). (c) qRT-PCR analysis of the expression of the indicated genes in the colon from Usp25+/+ (n = 5) and Usp25-/- (n = 5) mice after AOM/DSS induction for 9 weeks. (d-f) qRT-PCR analysis of the expression of the indicated genes in the colon tumors from Usp25+/+ (n = 5) and Usp25-/- (n = 5) mice after AOM/DSS induction (d), Vil-Cre;Trp53fl/fl (n = 6) and Vil-Cre;Trp53fl/flUsp25-/- (n = 6) mice at the 18th week after the first AOM injection (e), or ApcMin/+ (n = 5) and ApcMin/+Usp25-/- (n = 5) mice at 5-month-old (f). (g) qRT-PCR of the Wnt signaling genes in the Usp25+/+ (n = 3, technical replicates) and Usp25-/- (n = 3, technical replicates) colon organoids stimulated with Wnt3a (50 ng/ml) for 4 h. (h) Immunoblot analysis of the indicated proteins in Usp25+/+ (n = 3, technical replicates) and Usp25-/- (n = 3, technical replicates) colon organoids that were left untreated or stimulated with Wnt3a (50 ng/ml) for 4 h. (i) IHC images and the intensity quantification of Axin2, SOCS3, pStat3 and TNKS proteins in small intestine tumors from ApcMin/+ (n = 6) and ApcMin/+Usp25-/- (n = 6) mice at 5-month-old. (j, k) Immunoblot analysis and the intensity quantification of the indicated proteins in tumors from Usp25+/+ (n = 6) and Usp25-/- (n = 6) mice after AOM/DSS induction (j), or Vil-Cre;Trp53fl/fl (n = 5) and Vil-Cre;Trp53fl/flUsp25-/- (n = 5) mice at the 18th week after the first AOM injection (k). *P < 0.05; **P < 0.01; ***P < 0.001 (two-way ANOVA for g; two-tailed student’s t-test for c–f, i, j, k). Red and black scale bars represent 50 mm and 1 mm, respectively. Data are representative of at least three independent experiments (Graphs show mean in g or mean ± S.D. in c–f and i–k). Uncropped immunoblot images are shown in Uncropped_gels_Extended_Data_Fig.5. Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.5.

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Extended Data Fig. 6 Synthesis and toxicity analysis of AZ1.

(a, b) IHC images of USP25, b-Catenin, SOCS3 and pSTAT3 proteins (a) in patient CRC tissues (n = 36) and correlation analysis of USP25 with b-Catenin, SOCS3 and pSTAT3 proteins expression level (b). (c, d) The synthetic method (c) and HPLC chromatography (d) of the inhibitor AZ1. (e, f) Weight change (e) and images of spleens and lymph nodes (f) of 7-week-old male Usp25+/+ (n = 5) and Usp25-/- (n = 5) mice that were daily injected of PBS or AZ1 (40 mg/kg body weight) by gavage. Scale bars represent 0.4 mm. Data are representative of two experiments (a, e, f) (Graphs show mean ± S.D., two-tailed student’s t-test). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.6.

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Extended Data Fig. 7 Selectivity analysis of AZ1 on USP25.

(a, b) qRT-PCR analysis of the expression of the indicated genes in Usp25+/+ (n = 3, technical replicates) and Usp25-/- (n = 3, technical replicates) colon organoids infected with C. rodentium (a) or treated with Wnt3a (50 ng/ml) (b) in the presence or absence of AZ1 (10 mM) stimulation for 4 h. (c) Growth rate (as indicated by the OD600 values) of different bacteria that were inoculated to fresh LB culture medium (1:200) for the indicated time points after overnight culture (n = 3, technical replicates). (d) Shannon diversity (left), bacterial relative abundance (middle) and PCoA analysis from 16 S rRNA seq in feces of WT mice fed with PBS (n = 5 mice) or AZ1 (n = 5 mice) for 15 days. (e) Shannon diversity (left), bacterial relative abundance (middle) and PCoA analysis from 16 S rRNA seq in feces of WT mice treated by 2.5% DSS for 5 d followed by normal water for 2d in the presence of PBS (n = 4 mice) or AZ1 (n = 5 mice) by gavage. (f, g) Body weight change (f) and gross morphological change and lengths of colons (g) of Usp25-/- mice that were given 2.5% DSS in drinking water for 5 d, followed by normal drinking water for another 2 d in the presence or absence of seven successive daily gavage of PBS (n = 8 mice) or AZ1 (40 mg/kg) (n = 8 mice). (h, i) Weight change (h) and image and colon lengths (i) of Il10-/- mice (8-week old) housed in conventional open cage conditions and injected with PBS (n = 9 mice) or AZ1 (40 mg/kg) (n = 11 mice) by gavage every three days starting from 12-week old for 8 successive weeks. (j, k) qRT-PCR of the distal colon tissues (j) (n = 5 mice for PBS and AZ1 groups) and HE staining of colon (g) (PBS, n = 9 mice; AZ1, n = 11 mice) of Il10-/- mice treated as in (h). *P < 0.05; **P < 0.01; ***P < 0.001. (two-tailed Student’s t-test). Scale bars represent 300 μm. Data are representative of three (a–e) or two (j) independent experiments or combined two independent experiments (f–i) (Graphs show mean in a-c or mean ± S.D. in d-j). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.7.

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Extended Data Fig. 8 AZ1 has minimal effect on colon tumorigenesis in the USP25 deficient background.

(a) A scheme of colon tumor induction in Vil-Cre;Trp53fl/flUsp25-/- mice (upper). Representative images and tumor counts of colons from Vil-Cre;Trp53fl/flUsp25-/- mice that were weekly injected with AOM (10 mg/kg) for six successive weeks, followed by injection of PBS (n = 6 mice) or AZ1 (20 mg/kg) (n = 6 mice) by gavage every three days from 12th to 18th weeks after initial AOM injection (lower). (b) qRT-PCR of the indicated genes in colon tumors from Vil-Cre;Trp53fl/flUsp25-/- mice treated in (a) (n = 3 mice for PBS and AZ1 groups). (c) A model for USP25 to modulate colonic infections, inflammations and colon cancer. USP25 inhibits the expression of anti-bacteria genes, promotes bacterial infections and inflammation in the colon, downregulates SOCS3 to facilitate activation of STAT3, and promotes Wnt signaling to increase the stemness of colonic epithelial cells, which are inducers of colon cancer. Pharmacologically inhibition of USP25 impairs inflammation, restricts bacterial infections and inhibits tumorigenesis in the colon. Two-tailed student’s t-test. Data are combination (a) or representative (b) of two independent experiments (Graph show mean ± S.D.). Numerical source data for the experiments in this figure can be found in Numerical_Source_Data_Extended_Data_Fig.8.

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Wang, XM., Yang, C., Zhao, Y. et al. The deubiquitinase USP25 supports colonic inflammation and bacterial infection and promotes colorectal cancer. Nat Cancer 1, 811–825 (2020). https://doi.org/10.1038/s43018-020-0089-4

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