Intestinal inflammation targets cancer-inducing activity of the microbiota

Abstract

Inflammation alters host physiology to promote cancer, as seen in colitis-associated colorectal cancer (CRC). Here, we identify the intestinal microbiota as a target of inflammation that affects the progression of CRC. High-throughput sequencing revealed that inflammation modifies gut microbial composition in colitis-susceptible interleukin-10-deficient (Il10(-/-)) mice. Monocolonization with the commensal Escherichia coli NC101 promoted invasive carcinoma in azoxymethane (AOM)-treated Il10(-/-) mice. Deletion of the polyketide synthase (pks) genotoxic island from E. coli NC101 decreased tumor multiplicity and invasion in AOM/Il10(-/-) mice, without altering intestinal inflammation. Mucosa-associated pks(+) E. coli were found in a significantly high percentage of inflammatory bowel disease and CRC patients. This suggests that in mice, colitis can promote tumorigenesis by altering microbial composition and inducing the expansion of microorganisms with genotoxic capabilities.

Figures

Fig 1

Inflammation alters fecal microbial community structure. (A–B) Luminal microbiota of Il10−/− vs. WT mice. (A) Operational taxonomic unit (OTU) abundances were standardized by total, square root transformed, and assembled into a Bray Curtis similarity matrix to generate a multidimensional scaling (MDS) plot, where in these plots each symbol represents the microbiota of an individual mouse analyzed by Illumina sequencing of 16S V6 region. Il10−/− vs. WT comparison by ANOSIM (Analysis of Similarity), R=1 is maximum dissimilarity. (B) Richness, mean + SEM of cage means, 5–6 cages/group, 2–4 mice per cage, t test. (C–D) Luminal microbiota of AOM/Il10−/− vs. Il10−/− (C) MDS plot, AOM/Il10−/− vs. Il10−/−comparison by ANOSIM. (D) Richness, mean + SEM of cage means, 2–3 cages/group, 2–4 mice per cage, t test. (E–G) Standardized transformed abundance, median + SEM of cage means, 5–6 cages/group, 2–4 mice per cage, Mann Whitney U. (H) MDS plot depicting luminal microbiota of Il10−/− vs. WT, overlaid with Enterobacteriaceae abundance depicted by circle size. (I) E. coli ΔCt relative to total bacteria (16S). Each symbol depicts one mouse, line at median, Mann Whitney U.

Fig 2

Colonization of germ-free mice Il10−/− mice with E. coli (E.c.) or E. faecalis (E.f.) differentially impacts tumorigenesis without affecting inflammation. (A) Histologic inflammation scores, t test. (B) Macroscopic tumor counts; two tailed Mann Whitney. (C) Percent of mice with invasive adenocarcinoma; Fisher’s exact test. (D) Representative H&E histology. (A–D): Mean + SEM, 7–12/group, single experiment. (E) Colonic cytokine mRNA expression relative to GF Il10−/−; Mean + SEM, 4/group, t test.

Fig 3

Pks+ E. coli strains are associated with CRC and DNA damage. (A–B) PCR screen for pks (A) using primers targeting the left (1824bp) and right (1413bp) ends of the pks island in E. coli strains NC101 and K12, and E. faecalis (E.f.), and (B) using primers targeting the right end and ClbB gene of the pks island in mucosa-associated E. coli isolated from human colorectal tissue specimens, Binomial test *p<0.05, ***p<0.001. (C–D) NC101 pks induces (C) γH2AX (MOI 20, 4hr) in IEC-6 cells; Mean + SEM, ANOVA + Tukey, and (D) G2 cell cycle arrest (MOI 100, 24hr). A, C–D are representative of 3 experiments.

Fig 4

Deletion of pks reduces the tumorigenicity, but not inflammatory potential, of E. coli NC101 in AOM/Il10−/− mice. (A) Histologic inflammation and (B) neoplasia scores in 3 cohorts of mice at weeks 12, 14+AOM, 18+AOM, (C–F) 18wks+AOM: (C) Histologic invasion score, (D) Macroscopic tumor number, (E) Mean macroscopic tumor diameter in each mouse (F) Representative H&E histology. (A–F) Each symbol represents data from one mouse, line at mean, pairwise comparisons by t test. (G) Cells/crypt with ≥4 γH2AX foci, and γH2AX IHC (400x). Each symbol represents data from mouse, ANOVA + Tukey, line at mean, arrowheads indicate γfoci+ cells.