IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

doi:10.1038/s41467-020-19627-7

. 2020 Nov 13;11(1):5762.

doi: 10.1038/s41467-020-19627-7.

IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

Miao Tian^#¹, Xiumei Wang^#¹, Jihong Sun^#², Wenlong Lin¹, Lumin Chen², Shengduo Liu³, Ximei Wu⁴, Liyun Shi⁵, Pinglong Xu³, Xiujun Cai⁶, Xiaojian Wang⁷

Affiliations

¹ Institute of Immunology and Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003, Hangzhou, China.
² Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 310016, Hangzhou, China.
³ The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, 310058, Hangzhou, China.
⁴ Department of Pharmacology, School of Medicine, Zhejiang University, 310058, Hangzhou, Zhejiang, China.
⁵ Department of Immunology and Medical Microbiology, Nanjing University of Chinese Medicine, 210046, Nanjing, China.
⁶ Department of General Surgery, Innovation Center for Minimally Invasive Techniques and Devices, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 310016, Hangzhou, China. srrsh_cxj@zju.edu.cn.
⁷ Institute of Immunology and Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003, Hangzhou, China. wangxiaojian@cad.zju.edu.cn.

^# Contributed equally.

PMID: 33188184
PMCID: PMC7666182
DOI: 10.1038/s41467-020-19627-7

IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

Miao Tian et al. Nat Commun. 2020.

. 2020 Nov 13;11(1):5762.

doi: 10.1038/s41467-020-19627-7.

Authors

Miao Tian^#¹, Xiumei Wang^#¹, Jihong Sun^#², Wenlong Lin¹, Lumin Chen², Shengduo Liu³, Ximei Wu⁴, Liyun Shi⁵, Pinglong Xu³, Xiujun Cai⁶, Xiaojian Wang⁷

Affiliations

¹ Institute of Immunology and Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003, Hangzhou, China.
² Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 310016, Hangzhou, China.
³ The MOE Key Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, 310058, Hangzhou, China.
⁴ Department of Pharmacology, School of Medicine, Zhejiang University, 310058, Hangzhou, Zhejiang, China.
⁵ Department of Immunology and Medical Microbiology, Nanjing University of Chinese Medicine, 210046, Nanjing, China.
⁶ Department of General Surgery, Innovation Center for Minimally Invasive Techniques and Devices, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, 310016, Hangzhou, China. srrsh_cxj@zju.edu.cn.
⁷ Institute of Immunology and Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003, Hangzhou, China. wangxiaojian@cad.zju.edu.cn.

^# Contributed equally.

PMID: 33188184
PMCID: PMC7666182
DOI: 10.1038/s41467-020-19627-7

Erratum in

Author Correction: IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin.
Tian M, Wang X, Sun J, Lin W, Chen L, Liu S, Wu X, Shi L, Xu P, Cai X, Wang X. Tian M, et al. Nat Commun. 2023 Nov 29;14(1):7862. doi: 10.1038/s41467-023-43761-7. Nat Commun. 2023. PMID: 38030635 Free PMC article. No abstract available.

Abstract

Occurrence of Colorectal cancer (CRC) is relevant with gut microbiota. However, role of IRF3, a key signaling mediator in innate immune sensing, has been barely investigated in CRC. Here, we unexpectedly found that the IRF3 deficient mice are hyper-susceptible to the development of intestinal tumor in AOM/DSS and Apc^min/+ models. Genetic ablation of IRF3 profoundly promotes the proliferation of intestinal epithelial cells via aberrantly activating Wnt signaling. Mechanically, IRF3 in resting state robustly associates with the active β-catenin in the cytoplasm, thus preventing its nuclear translocation and cell proliferation, which can be relieved upon microbe-induced activation of IRF3. In accordance, the survival of CRC is clinically correlated with the expression level of IRF3. Therefore, our study identifies IRF3 as a negative regulator of the Wnt/β-catenin pathway and a potential prognosis marker for Wnt-related tumorigenesis, and describes an intriguing link between gut microbiota and CRC via the IRF3-β-catenin axis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. IRF3 in intestinal epithelium protects against colonic tumorigenesis.**
a Representative images of colon tumors from IRF3^+/+ and IRF3^−/− mice on day 90 after AOM/DSS model. b–d Colon tumor counts, size, and tumor load from IRF3^+/+ and IRF3^−/− mice (n = 15 mice/group) after AOM/DSS model (day 90). e Representative images of the small intestine and tumors in Apc^min/+ and Apc^min/+IRF3^−/− mice. f–h Intestinal tumors counts, size, and tumor load from Apc^min/+ and Apc^min/+IRF3^−/− mice (n = 15 mice/group). i–l Three groups of mice were generated by bone marrow transplantation: IRF3^+/+ → IRF3^+/+, n = 10; IRF3^−/− → IRF3^+/+, n = 10; IRF3^+/+ → IRF3^−/−, n = 8; the numbers and size of tumors in the colon were quantified after AOM/DSS model (day 90). m–p Colon tumor counts, size, and tumor load from IRF3^fl/fl and IRF3^fl/fl Villin^cre mice (n = 13 mice/group) representative images of colons at left (m) after AOM/DSS model (day 90). Each symbol represents one mouse (b–d, f–h, j–l, n–p). *P < 0.05; **P < 0.01; ***P < 0.001; NS not statistically significant by two-tailed t test (b–d, f–h, j–l, n–p). Data are from two independent experiments (a–p) and are presented as mean ± s.e.m. in b–d, f–h, j–l, n–p. See also Supplementary Fig. S1.

**Fig. 2. Deficiency of IRF3 promotes the proliferation of intestinal epithelial cells.**
a Standardized Ki67 immunostaining of the distal colon, and tumors from IRF3^+/+ and IRF3^−/− mice on day 0, 15, and 90 after AOM injection. Scale bar, 100 μm. b Quantification of the number of Ki67⁺ in each crypt from IRF3^+/+ and IRF3^−/− mice (day 0, n = 3; day 15, n = 5; day 90, n = 5). c Standardized Ki67 immunostaining of the distal colon and tumors from IRF3^fl/fl and IRF3^fl/flVillin^cre mice on day 0, 15, and 90 after AOM injection. Scale bar, 100 μm. d Quantification of the number of Ki67⁺ in each crypt from IRF3^fl/fl and IRF3^fl/flVillin^cre mice (day 0, n = 3; day 15, n = 4; day 90, n = 5). e Standardized Ki67 immunostaining of the distal colon and tumors from chimera on day 90 after AOM injection. Scale bar, 100 μm. f Quantification of the number of Ki67⁺ in each crypt of chimera mice (day 90, n = 3 mice/group). *P < 0.05; **P < 0.01; ***P < 0.001; NS not statistically significant by two-tailed t test (a–f). Data represent two independent experiments (a–f) and are presented as mean ± s.e.m. in a–f. See also Supplementary Fig. S2.

**Fig. 3. IRF3 suppresses the CRC via inhibiting Wnt signaling.**
a The signal pathways were enriched with the 65 genes that upregulated in tumor tissue only in “KO” from the RNA-seq analysis results. b Immunofluorescence analysis of β-catenin nuclear translocation in colorectal tumors from IRF3^+/+ and IRF3^−/− mice after treatment with AOM/DSS (days 0 and 90). Scale bar, 20 μm. c, d Real time qPCR analysis for expression of the Wnt target, and associated genes in the distal colon and tumors from IRF3^fl/fl and IRF3^fl/flVillin^cre (day 0, n = 3 mice/group; day 15, n = 4 mice/group; day 90, n = 7 mice/group) mice. e–f Images (e) and quantifications (f) of the number (left) and size (right) of organoids from IRF3^+/+ and IRF3^−/− colon stem cells. g Representative images of colon tumors from IRF3^fl/fl and IRF3^fl/flVillin^cre mice on day 90 after AOM/DSS model with pbs or ICG-001 treatment. h–j Colon tumors counts, size, and tumor load in AOM/DSS-treated mice with PBS or ICG-001 treatment (300 mg/kg per day, orally, once daily, six times 1 week for the last 10 weeks of the AOM/DSS model; PBS group, n = 6 mice/group; ICG-001 group, n = 7 mice/group). k Representative MRI images of IRF3^fl/fl and IRF3^fl/flVillin^cre mice with PBS or ICG-001 treatment (300 mg/kg per day, orally, once daily, six times 1 week for the last 10 weeks of the AOM/DSS model). Arrowhead indicates colon tumor. Each symbol represents one organoid (e) or an individual mouse (c, d, h–j). *P < 0.05; **P < 0.01; ***P < 0.001; NS not statistically significant by two-tailed t test (c–f, h–j). Data represent two (b–d, g–k) or three independent experiments (e, f), and are presented as mean ± s.e.m. in a–j. See also Supplementary Fig. S3.

**Fig. 4. The cytoplasmic IRF3 in resting state inhibits the cell proliferation and Wnt/β-catenin pathway in HCT116 and H1299 cell lines.**
a, b Proliferation of the IRF3^+/+ and IRF3^−/− HCT116 (a) and H1299 (b) cells. c Colony formation experiment of the IRF3^+/+ and IRF3^−/− HCT116 and H1299 cells. d Proliferation of the HCT116 cells with Wnt signaling inhibitor ICG-001 (50 μM) or DMSO treatment. e, f Representative images of tumors from subcutaneous tumor formation assay in nude mice (e). Subcutaneous tumor formation assay in nude mice with 2 × 10⁶ IRF3^+/+ or IRF3^−/− HCT116 cells per mouse. After 1 week of the injection, PBS or ICG-001 treatment (200 mg/kg, i.v., once daily) was applied in mice until the end of the model. Tumor weight for each group (n = 4) was plotted (f) at day 21 after injection. g Immunohistochemical analysis for ki67 in tumors from e. h Proliferation of β-catenin-wild type (Ctnnb1^+/+) and β-catenin-knockout (Ctnnb1^−/−) HCT116 cells treated with siNC or siIRF3. i, j Proliferation of the IRF3^+/+ and IRF3^−/− HCT116 (i) and H1299 (j) cells transfected with the indicated plasmids expressing backbone, IRF3, IRF3-ΔnDB, IRF3-ΔNLS, or IRF3-5D. k Real time qPCR analysis for the Wnt target, and associated genes in IRF3^+/+ and IRF3^−/− HCT116 cells transfected with the indicated plasmids expressing backbone, IRF3, IRF3-ΔnDB, IRF3-ΔNLS, or IRF3-5D. l, m The stable expression control plasmid, Flag-tagged IRF3, -IRF3-ΔnDB, -IRF3-ΔNLS, and IRF3-5D mutations IRF3^+/+ or IRF3^−/− HCT116 cells were applied to subcutaneous tumor formation assay in nude mice with 2 × 10⁶ cells/group per mouse for 21 days. Images of tumor grafts from these cells at day 21 (l). Tumor weight for each group (n = 4) was plotted in m. Each symbol represents one mouse (m). *P < 0.05; **P < 0.01; ***P < 0.001; NS not statistically significant by two-tailed t test (a–m). Data represent two (e–g, l, m) or three independent experiments (a–d, h–k) and are presented as mean ± s.e.m. in a–m. See also Supplementary Fig. S4.

**Fig. 5. IRF3 binds the ARM domain of β-catenin and prevents its nucleus translocation.**
a, b Nucleocytoplasmic separation and immunoblot analysis of Active-β-catenin in HCT116 (a) and H1299 (b) cells after treated with wnt3a-conditioned medium. c Immunoblot analysis of the endogenous interaction between active-β-catenin, β-catenin, or GSK3β and IRF3 with anti-IRF3 immunoprecipitates in HCT116 cell line extracts after treated with wnt3a-conditioned medium. d Immunoblot analysis of the interaction between β-catenin or β-catenin-S33A and IRF3 with anti-FLAG immunoprecipitates in HEK293T cell line. e Pull-down analysis the interaction between GST-β-catenin, GST-β-catenin-ARM, or GST-β-catenin-Δ634-663 and MBP-IRF3. f, g Immunofluorescence (f) and nucleocytoplasmic separation (g) analysis for the cellular localization of β-catenin or its mutants in HEK293 cell line upon wnt3a-conditioned medium treatment. Red scale bars, 10 μm. Data represent three independent experiments (a–g). Source data are provided as a Source data file. See also Supplementary Fig. S5.

**Fig. 6. Activation of IRF3 by PRR signaling relieves its inhibition on Wnt signaling.**
a TOPflash-relative luciferase activity analysis for VSV treatment in IRF3-knockout HCT116 cells. b Nucleocytoplasmic separation and immunoblot analysis of active-β-catenin (active-β-cat.) and IRF3 activation in HCT116 cas9 cells after treated with VSV. c TOPflash-relative luciferase activity analysis for VSV treatment in siNC and siIRF3 HCT116 cells. d Immunoblot analysis for the interaction between IRF3, IRF3-ΔnDB, IRF3-ΔNLS, or IRF3-5D and β-catenin with anti-FLAG immunoprecipitates of HEK293T cells. e Immunoblot analysis for the interaction between IRF3 or IRF3-5D and β-catenin-S33A with anti-FLAG immunoprecipitates in HEK293T cells. f Immunoblot analysis for the endogenous interaction between active-β-catenin or β-catenin and IRF3 with anti-IRF3 immunoprecipitates in HCT116 cell line extracts treated with VSV. g Representative images of the small intestine tumors from 5-month-old Apc^min/+ and Apc^min/+ IRF3^−/− mice with 4 months Abx treatment. h The small intestine tumor counts from Apc^min/+ and Apc^min/+ IRF3^−/− mice with Abx treatment (n = 10, n = 11, n = 10, n = 10). i Standardized TCF1 and MX1 immunostaining of the small intestine and tumors from Apc^min/+ and Apc^min/+ IRF3^−/− mice with Abx treatment or without Abx treatment. Scale bars, 50 μm. Each symbol represents one mouse (h). *P < 0.05; **P < 0.01; ***P < 0.001; NS not statistically significant by two-tailed t test (a, c, h). Data are from two (g–i) or three (a–f) independent experiments and are presented as mean ± s.e.m. in a, c, h. Source data are provided as a Source data file. See also Supplementary Fig. S6.

**Fig. 7. IRF3 expression correlates with the activation of Wnt signaling and the survival of CRC, lung adenocarcinoma, and hepatocellular carcinoma patients.**
a, b Correlation analysis for IRF3, LEF1, and TCF1 expression in CRC patients (n = 115) (a) or in human lung carcinomas (n = 67) (b). Fisher’s exact test. c, d Kaplan–Meier analysis for overall survival in a set of CRC patients (c) or human lung carcinomas (d) according to IRF3, LEF1, and TCF1 expression. e, f Combined expression status of IRF3, LEF1, and TCF1 in a set of CRC patients (e) or human lung carcinomas (f). Log-rank test, log rank, p < 0.0001. g Correlation between IRF3 expression and LEF1 or TCF1 expression in human hepatocellular carcinoma patients. n = 92 cases, Fisher’s exact test. h, i Kaplan–Meier analysis for the overall survival in a set of hepatocellular carcinoma patients according to IRF3, LEF, and TCF1 expression (h), or combined expression status of IRF3, LEF1, and TCF1 (i). *P < 0.05; **P < 0.01 by two-sided Pearson correlation coefficient (a, b, g). Log-rank test, log rank, P < 0.0001. *P < 0.05; **P < 0.01; ***P < 0.001 (c–f, h, i). See also Supplementary Fig. S7.

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References

1. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 2014;383:1490–1502. doi: 10.1016/S0140-6736(13)61649-9. - DOI - PubMed
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[1] Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 2014;383:1490–1502. doi: 10.1016/S0140-6736(13)61649-9. - DOI - PubMed

[2] Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 2014;383:1490–1502. doi: 10.1016/S0140-6736(13)61649-9. - DOI - PubMed

[3] Sjoblom T, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314:268–274. doi: 10.1126/science.1133427. - DOI - PubMed

[4] Sjoblom T, et al. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314:268–274. doi: 10.1126/science.1133427. - DOI - PubMed

[5] Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–205. doi: 10.1016/j.cell.2012.05.012. - DOI - PubMed

[6] Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–205. doi: 10.1016/j.cell.2012.05.012. - DOI - PubMed

[7] Shanahan F, et al. Feeding the microbiota: transducer of nutrient signals for the host. Gut. 2017;66:1709–1717. doi: 10.1136/gutjnl-2017-313872. - DOI - PubMed

[8] Shanahan F, et al. Feeding the microbiota: transducer of nutrient signals for the host. Gut. 2017;66:1709–1717. doi: 10.1136/gutjnl-2017-313872. - DOI - PubMed

[9] Kostic AD, et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe. 2013;14:207–215. doi: 10.1016/j.chom.2013.07.007. - DOI - PMC - PubMed

[10] Kostic AD, et al. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe. 2013;14:207–215. doi: 10.1016/j.chom.2013.07.007. - DOI - PMC - PubMed

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IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

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IRF3 prevents colorectal tumorigenesis via inhibiting the nuclear translocation of β-catenin

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