ZFP90 drives the initiation of colitis-associated colorectal cancer via a microbiota-dependent strategy

Abstract

Chronic inflammation and gut microbiota dysbiosis are risk factors for colorectal cancer. In clinical practice, patients with inflammatory bowel disease (IBD) have a greatly increased risk of developing colitis-associated colorectal cancer (CAC). However, the underlying mechanism of the initiation of CAC remains unknown. Systematic analyses using an existing genome-wide association study (GWAS) and conditional deletion of Zfp90 (encoding zinc finger protein 90 homolog) in a CAC mouse model indicated that Zfp90 is a putative oncogene in CAC development.Strikingly, depletion of the gut microbiota eliminated the tumorigenic effect of Zfp90 in the CAC mouse model. Moreover, fecal microbiota transplantation demonstrated that Zfp90 promoted CAC dependent on the gut microbiota. Analysis of 16s rDNA sequences in fecal specimens from the CAC mouse model allowed us to speculate that a Prevotella copri-defined microbiota might mediate the oncogenic role of Zfp90 in the development of CAC. Mechanistic studies revealed Zfp90 accelerated CAC development through the TLR4-PI3K-AKT-NF-κB pathway. Our findings revealed the crucial role of the Zfp90-microbiota-NF-κB axis in creating a tumor-promoting environment and suggested therapeutic targets for CAC prevention and treatment.

Keywords: ZFP90; colitis-associated colorectal cancer; gut barrier; gut microbiota; prevotella copri.

Figure 1.

ZFP90 is associated with CRC and IBD and Zfp90^ΔIEC mice are less susceptible to AOM-DSS-induced CAC a Venn diagram showing the chromosome loci accounting for CRC (46 loci), IBD (166 loci), and both (16 loci). b Venn diagram showing the genes regulated by CRC-related SNPs (56 genes), IBD-related SNPs (209 genes), and both (13 genes). c ZFP90 was identified after overlapping 13 candidate target genes with genes differentially expressed during AOM-DSS treatment (918 genes). d The body weight of Zfp90^fl/fl and Zfp90^ΔIEC mice was recorded throughout the experiment and was expressed as the ratio relative to the initial weight before DSS treatment (n = 8 per group). e Representative images of distal and middle colons in AOM-DSS-treated Zfp90^fl/fl and Zfp90^ΔIEC mice. f Tumor distribution in Zfp90^fl/fl and Zfp90^ΔIEC mice (n = 8 per group). g Colon length in Zfp90^fl/fl and Zfp90^ΔIEC mice at the end of the experiment (n = 8 per group). h Representative H&E staining of colon tumors from Zfp90^fl/fl and Zfp90^ΔIEC mice. * tumors. i Proportion of low-grade dysplasia, high-grade dysplasia, and adenocarcinoma in the colons. j Representative immunofluorescence staining of the PCNA protein in tumor tissues from Zfp90^fl/fl and Zfp90^ΔIEC mice. Sections were stained with DAPI (blue), CDH1 (green) and PCNA (red). k The percentage of PCNA-positive tumor cells was quantified (n = 5 per group). l Representative immunofluorescence staining of C-CAS3-positive cells in tumor tissues from Zfp90^fl/fl and Zfp90^ΔIEC mice. Sections were stained with DAPI (blue), CDH1 (green) and C-CAS3 (red). m The percentage of C-CAS3-positive tumor cells was quantified (n = 5 per group). Data with error bars represent the mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. Two-way ANOVA (d, f), nonpaired two-tailed t-test (g, k, m), Fisher’s exact test (i)

Figure 2.

Zfp90 deletion in IECs improves the gut barrier and alleviates local inflammation during CAC development

Figure 3.

The gut microbiota is indispensable for the antitumor phenotype in Zfp90^ΔIEC mice. a Representative images of colons in antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice after AOM-DSS treatment. b Tumor numbers in antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice (n = 7 per group). c Representative immunofluorescence staining of the PCNA protein in tumor tissues from antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice after AOM-DSS treatment. Sections were stained with DAPI (blue), CDH1 (green) and PCNA (red). d The percentage of PCNA-positive tumor cells was quantified (n = 5 per group). e Representative immunofluorescence staining of C-CAS3 protein in tumor tissues from antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice after AOM-DSS treatment. Sections were stained with DAPI (blue), CDH1 (green) and C-CAS3 (red). f The percentage of C-CAS3-positive tumor cells was quantified (n = 5 per group). g Intestinal permeability assessed by FITC-dextran in antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice (n = 5 per group). h, i Real-time PCR was performed to determine the mRNA expression of Il17a and Il1b in the colonic epithelium from antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice (n = 5 per group). j Representative immunohistochemical staining for LY6G and F4/80 in colon segments from antibiotic-treated Zfp90^fl/fl and Zfp90^ΔIEC mice with AOM-DSS treatment. k-t Gavage of WT mice with feces from Zfp90^fl/fl and Zfp90^ΔIEC mice (n = 5–8 per group). Representative images of distal and middle colons (k), number of tumors (l), representative immunofluorescence staining of PCNA (m), percentage of PCNA-positive tumor cells (n), representative immunofluorescence staining of C-CAS3 (o), percentage of C-CAS3-positive tumor cells (p), the serum FITC-Dextran level (q), mRNA expression of Il17a and Il1b in the colonic epithelium (r, s), and representative immunohistochemical staining for LY6G and F4/80 (t) from WT (Zfp90^fl/fl mice) and WT (Zfp90^ΔIEC mice) under AOM-DSS treatment. Sections were stained with DAPI (blue), CDH1 (green) and PCNA or C-CAS3 (red). Data with error bars represent the mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. Nonpaired two-tailed t-test (b, d, f, g, h, i, l, n, p, r, s) and the Mann–Whitney U test (q) were used

Figure 4.

The gut microbiota is altered by intestine-specific deletion of oncogene Zfp90. a Principal component analysis plot based on bacterial 16S ribosomal DNA gene sequences of the fecal content from Zfp90^fl/fl and Zfp90^ΔIEC mice before and after AOM-DSS-treatment (n = 5 per group). b Differential abundances of bacteria in the fecal content of Zfp90^fl/fl and Zfp90^ΔIEC mice before and after AOM-DSS-treatment. c Real-time PCR was performed to determine the abundance of P. copri in the fecal content of Zfp90^fl/fl and Zfp90^ΔIEC mice before and after AOM-DSS-treatment. d Representative images of colons in PBS- and P. copri-treated WT mice after AOM-DSS treatment. e Tumor numbers in PBS- and P. copri-treated WT mice after AOM-DSS treatment. f Representative immunofluorescence staining of the PCNA protein in tumor tissues from PBS- and P. copri-treated WT mice after AOM-DSS treatment. Sections were stained with DAPI (blue), CDH1 (green) and PCNA (red). g The percentage of PCNA-positive tumor cells was quantified. h Representative immunofluorescence staining of C-CAS3 protein in tumor tissues from PBS- and P. copri-treated WT mice after AOM-DSS treatment. Sections were stained with DAPI (blue), CDH1 (green) and C-CAS3 (red). i The percentage of C-CAS3-positive tumor cells was quantified. j The correlation between P. copri abundance and ZFP90 expression in specimens from patients with CRC. Statistical analysis was performed using nonpaired two-tailed t-test (e, g, i), one-way ANOVA (c), and Wilcoxon’s rank-sum test (j)

Figure 5.

Decreased TLR4-dependent PI3K/AKT/NF-κB signaling in colon tissue of Zfp90^ΔIEC mice