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. 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 Lin  1 , Lumin Chen  2 , Shengduo Liu  3 , Ximei Wu  4 , Liyun Shi  5 , Pinglong Xu  3 , Xiujun Cai  6 , Xiaojian Wang  7
Affiliations

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

Miao Tian et al. Nat Commun. .

Erratum in

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 Apcmin/+ 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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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 Apcmin/+ and Apcmin/+IRF3−/− mice. f–h Intestinal tumors counts, size, and tumor load from Apcmin/+ and Apcmin/+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 IRF3fl/fl and IRF3fl/fl Villincre 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
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 IRF3fl/fl and IRF3fl/flVillincre 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 IRF3fl/fl and IRF3fl/flVillincre 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
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 IRF3fl/fl and IRF3fl/flVillincre (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 IRF3fl/fl and IRF3fl/flVillincre 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 IRF3fl/fl and IRF3fl/flVillincre 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
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
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
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 Apcmin/+ and Apcmin/+ IRF3−/− mice with 4 months Abx treatment. h The small intestine tumor counts from Apcmin/+ and Apcmin/+ 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 Apcmin/+ and Apcmin/+ 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
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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