The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors

Abstract

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as "pks" encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks⁺E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Keywords: Colibactin; Colorectal cancer; Escherichia coli; Mismatch repair; Mutation.

Fig. 1

NC101 colonization increases the number of somatic mutations and elicits a MMRd-associated signature in germ-free Apc^Min/+/DSS mice. a, Representative pictures of mouse colon tumors. Scale bars, 1 cm. b, H&E-stained colon Swiss roll sections (distal). Scale bars, 500 µM. c, Macroscopic tumor counts from Apc^Min/+/DSS mice colonized by NC101 or ΔclbP, n=14 (ΔclbP) and n=17 (NC101). Data are pooled from two individual experiments. d, spectra of somatic SBS mutations in Apc^Min/+/DSS mice colonized by NC101 or ΔclbP, the bottom panel shows NC101-associated mutations obtained by subtracting the relative mutational frequency in NC101-colonized mice from those in ΔclbP -colonized mice. e, Quantification of the total number of somatic SNPs in tumors from mice colonized by NC101 or ΔclbP. f, Relative contributions of known mutational signatures in somatic SBS profiles from individual mice colonized by NC101 or ΔclbP, n=6 per group. g, Relative contributions of the MMRd-associated mutational signature SBS15 in tumors from mice colonized by NC101 or ΔclbP, n=6 per group.

Fig. 2

Colibactin 742 causes cyclopropane-mediated DNA damage. a, Representative images of IEC-6 cells incubated with the MMC (3 µM), colibactin 742, or colibactin 746 at the indicated concentration for 24 hours before visualizing DNA strand breaks by immunofluorescent staining using γH2AX antibodies. Scale bar, 100 µm. b, The percentage of cells with >5 γH2AX foci from as shown in a were quantified. n=3, with each datapoint representing the average of 3-6 40x fields of view from a single independent experiment. c, Human colonic organoids were cultured for 48 hours before treatment with MMC, 742, or 746 at the indicated concentration for 48 hours before visualizing DNA strand breaks by immunofluorescent staining using γH2AX antibodies. Scale bar, 100 µm. d, The percentage of cells per organoid with >5 γH2AX foci from 3-5 colonoids per group as shown in c were quantified. Data are representative of 2 independent experiments. e, Representative images of human colonoids cultured for 48 hours in the presence of 742 or 746 at the indicated concentration. Scale bar, 200 µm. f, Quantification of colonoid diameter in e, with each data point representing the average of 30 colonoids. Data are pooled from three independent experiments. g, Quantification of the relative number of viable colonoids normalized to untreated 48 hours after seeding, categorized as those with approximate diameter >30 µm and debris absent from the organoid lumen, as visualized in e, with each data point representing the normalized colonoid count from a single experimental well. Data are pooled from three independent experiments.

Fig. 3

742 treatment activates p53 signaling and ER stress pathways in normal intestinal epithelial cells. a, Principal components analysis of global transcriptomic data from cells treated with colibactin 742 or colibactin 746 at the indicated time and concentration. b-e, Volcano plot of differential gene expressions in 742:746 comparisons after 4-hour 10 µM (b), 4-hour 100 µM (c), 12-hour 10 µM (d), or 12-hour 100 µM (e), genes with FDR < 0.05 enrichment in each treated denoted by color. f-g IPA analysis of significantly enriched pathways in FHC cells treated with 10 µM 742 (f) or 100 µM 742 (g) for 12 hours, relative to 746 treated cells. h-i, Top 10 activated and inhibited upstream regulators with p-value < 0.05 after in FHC cells treated with 10 µM 742 (h) or 100 µM 742 (i) for 12 hours, relative to 746 treated cells. j, Heatmap of select genes significantly enriched (FDR<0.05) in at least one condition and identified as a component of significantly enriched pathways in f,g.

Fig. 4

Chronic 742 exposure upregulates BRCA1 and FA associated DNA repair pathways. a, Diagrammatic representation of experimental procedure for chronic colibactin 742 or colibactin 746 treatment and derivation of subclones for RNAseq and WGS analysis. b, Principal components analysis of global transcriptomic data from sub-clonal HCT 116 cells treated with 20 µM 742 or 746 for ten 48-hour cycles. c, Volcano plot showing differential gene expressions in after chronic exposure to 742 or 746 in sub-clonal HCT 116 cells, genes with FDR < 0.05 enrichment in each treated denoted by color. d-e, Analysis of significantly enriched pathways in sub-clonal chronic 742-treated HCT 116 cells as shown in a by IPA (d) or PID (e), relative to sub-clonal chronic 746-treated cells. f, Top 10 activated and inhibited upstream regulators with p-value < 0.05 after in sub-clonal chronic 742-treated HCT 116 cells as shown in a, relative to sub-clonal chronic 746-treated cells. g, Pre-ranked GSEA enrichment profile of KEGG mismatch repair and homologous recombination pathways in sub-clonal chronic 742-treated HCT 116 cells, relative to sub-clonal chronic 746-treated cells.

Fig. 5

742 increases T>N SBS and pks-associated ID signatures in intestinal epithelial cells. a, Quantification of the total SNVs in sub-clonal HCT 116 cells treated with 20 µM colibactin 742 or colibactin 746 for ten 48-hour cycles. b-c, the percentage of T>N (b) and [T>N]T (c) single base substitutions (SBS) relative to the total number of observed substitutions in sub-clonal HCT 116 cells after chronic treatment treated with 20 µM 742 or 746. d, spectra of SBS mutations in sub-clonal HCT 116 cells after chronic treatment with 20 µM 742 or 746, with 742-associated mutations obtained by subtracting the absolute mutational frequency in 742-treated cells from those in 746-treated cells. Data is shown as the proportion of total SBS. e, decomposition of the subtracted (742-746) spectra decomposed into five constituent known signatures, and the relative contribution of constituent signatures. f, Quantification of the total indels (ID) in sub-clonal HCT 116 cells treated with 20 µM colibactin 742 or colibactin 746 for ten 48-hour cycles. g, spectra of short (<6bp) ID mutations in sub-clonal HCT 116 cells after chronic treatment with 20 µM 742 or 746, with 742-associated mutations obtained by subtracting the absolute mutational frequency in 742-treated cells from those in 746-treated cells. Data is shown as the relative proportion of total short IDs. h, relative contribution of known ID signatures in 742-treated cells; the number of samples with extracted signature and the contribution quantified as indels per Mb is shown with the median contribution denoted by a red line. i, co-occurrence rate of COSMIC SBS signatures with a proposed etiology and SBS88 in 2945 previously analyzed CRC tumor tissue samples. j, total number of SBS44 mutations in patients with or without the co-occurrence of SBS88 (n=358).