Genomic aberrations after short-term exposure to colibactin-producing E. coli transform primary colon epithelial cells

Abstract

Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin’s distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. The genetic landscape of mutations that drive carcinogenesis is well explored and involves the Wnt, KRAS, TGF-β, and p53 pathways¹. Combinations of mutations in these pathways are responsible for the transformation of epithelial cells^2,3, and constitutively active Wnt signaling is considered to be a crucial feature of almost all CRCs⁴. Wnt signaling drives proliferation of colon cells and is physiologically active only in the stem cell compartment, while differentiated cells lack Wnt signaling⁵. Experimental and patient-derived data indicate that constitutively active Wnt signaling is an early event in the CRC cascade, often achieved by mutation of APC and subsequent loss of heterozygosity (LOH)^1,6. Although, this holds true for sporadic cases of CRC, cases driven by chronic inflammatory disorders, such as ulcerative colitis or Crohn’s colitis, are associated with a distinct mutational profile, in which mutations in the p53 pathway are thought to occur as an early event^7,8,9,10. While many of the mutations in driver genes are due to single nucleotide substitutions and small insertion and deletions, chromosomal aberrations are frequent in CRC and found in the majority of cases⁴. Chromosomal instability (CIN) is often attributed to cell-intrinsic causes like spindle assembly checkpoint defects¹¹, but the exact mechanisms leading to CIN are mostly unknown.

Recent data suggest that environmental factors are important drivers of CRC and that the colonic microbiota plays an important role in both the development and progression of CRC^12,13,14. In addition to microbial factors that induce inflammation and therefore contribute to carcinogenesis by triggering oxidative stress or changes in the stem cell niche, certain bacteria that colonize the gut appear to directly affect the genomic integrity of epithelial cells through genotoxins. In this context, colibactin is a prominent genotoxin produced by several Enterobacteriaceae members, such as E. coli belonging to phylogenetic group B2. This toxin is a polyketide/nonribosomal peptide hybrid, encoded by the pks pathogenicity island. Infection of eukaryotic cell lines with pks+ E. coli was shown to cause DNA double-strand breaks (DSBs), leading to megalocytosis and cell cycle arrest¹⁵. Short exposure of an intestinal epithelial cell line to pks+ E. coli has been reported to cause genomic instability and anchorage-independent growth¹⁶. Significant enrichment of pks gene clusters has been observed in CRC metagenomes¹³. An increase in colonic mucosa-associated E. coli that possess the pks gene has been observed in inflammatory bowel disease (IBD), familial adenomatous polyposis (FAP), and CRC patients^17,18,19. Colibactin alkylates DNA through covalent modifications and forms stable adenine-colibactin adducts in cultured mammalian cells and in vivo^20,21,22. It has also been shown to leave a specific mutational signature in CRC patients^23,24. Moreover, pks+ E. coli enhance tumorigenesis in different murine models of FAP and CRC, such as mice heterozygous for Apc¹⁸, interleukin-10–deficient (Il10^−/−) germ-free mice¹⁷, and the azoxymethane/dextran sulfate sodium (AOM/DSS) model²⁵. Notably, in these mouse models, tumors can also arise in the absence of colibactin, leaving it uncertain whether colibactin merely enhances tumorigenesis or whether it can also play a causative role. To study genomic instability caused by pks+ E. coli in vitro, infections have only been performed with transformed or immortalized cell lines, and it remains unclear whether healthy primary colon epithelial cells respond to infection in the same way.

To address this, we developed primary cell culture models derived from normal murine colon epithelial cells. We infected organoids as well as polarized monolayers derived from primary colon epithelial cells with pks+ E. coli or an isogenic mutant that is defective for colibactin synthesis. In these models, we were able to reproduce early responses to infection, including DSBs and development of megalocytosis—effects unique to strains with a functional pks island. Furthermore, we examined whether colibactin-induced DNA damage after short-term infection causes genomic instability, mutations, or changes in niche dependence of colon organoids. We specifically investigated Wnt-independence due to its clinical relevance. Our findings reveal that the genotoxic effect of colibactin is sufficient to rapidly cause genomic aberrations in primary colon cells and to induce multiple features of transformation reminiscent of CRC. These transformed cells did not exhibit the typical colibactin mutational signature, suggesting that improper repair of colibactin-induced cross-links after short exposure to the bacteria can lead to copy number variations (CNV) and other mutations. Accordingly, chromosomal aberrations causing aneuploidy induced by colibactin are sufficient to cause primary cell transformation and precede detectable genomic imprinting of the colibactin mutational signature. The transforming capacity of colibactin-producing E. coli may thus be higher than what is estimated from the presence of the mutational signature alone…