A bacterial mutational footprint in colorectal cancer genomes

Summary

Changes in the microbiome are associated with the development of colorectal cancer, but causal explanations have been lacking. We recently demonstrated that pks⁺ Escherichia coli induce a specific mutational pattern using intestinal organoids and these mutations are present in the genomes of colorectal cancer. This finding warrants further studies on the microbial role in oncogenic mutation induction, cancer development and future preventive strategies.

Main

Numerous bacteria of the intestinal microbiota have been associated with the development of colorectal cancer (CRC). Yet, demonstrating causal roles of individual bacterial species in the initiation of CRC remains a formidable challenge. New model systems such as organoids—miniature versions of healthy and diseased patient tissues—have recently emerged as tools to bridge this association-causation gap through mechanistic studies on host--microbe interactions.

Strains of genotoxic pks⁺ Escherichia coli are present in the microbiomes of an estimated 10–20% of people in the Western world and are enriched in faeces/gut of patients with CRC. The presence of the pks operon in the genome of some E. coli strains was discovered in 2006 and has been linked to the production of the genotoxin colibactin.¹ This polyketide can damage the DNA of eukaryotic cells¹ and promote tumorigenesis in CRC mouse models.^2,3 For these reasons, genotoxic E. coli are prime candidates to assess whether bacteria can induce carcinogenesis by enhancing mutation accumulation in intestinal cells.

Mutational signatures are characteristic patterns of single base substitutions (SBS) or short insertions and deletions (Indels) in the genomic DNA of cells/tumours, which reflect activity of endogenous mutagenic processes or exposure to environmental mutagens. Once a mutational signature has been linked to the action of a specific agent, it can be used to estimate the mutation burden induced by this mutagen in human tumours. Unique mutational signatures had been associated with a variety of mutagens, such as tobacco smoke or UV exposure⁴ but not with bacterial exposure.

By co-culturing human intestinal organoids with pks⁺ E. coli over a time frame of several months, we were able to identify two co-occurring mutational signatures, a single base substitution signature, SBS-pks and an indel signature, ID-pks (Fig. 1), which have been recently included in the COSMIC catalogue of mutational signatures as SBS88 and of ID18, respectively.⁵ These colibactin-induced signatures are characterised by thymine substitutions or deletions in a specific 5-base DNA motif that consists predominantly of adenine and thymine residues. The motif fits with the proposed model that colibactin alkylates adenines on opposite strands and thereby crosslinks DNA.^6,7 SBS88 and ID18 were concurrently present in approximately 2% to 7% of CRC patients from independent primary and metastatic CRC cohorts, respectively. Individual mutations matching the motifs of the colibactin signatures are contributing to cancer-driving mutations in key CRC genes such as adenomatous polyposis coli (APC) and the full impact of such mutations is only beginning to be unravelled by detailed cancer genome analyses.⁸ Such insights into common mutational outcomes of colibactin exposure may be invaluable in hereditary cancer settings such as familial adenomatous polyposis, where a germline mutation in APC leads to polyp formation early in life and enrichment of genotoxic E. coli has been observed.³

Fig. 1. pks⁺ mutational signatures in intestinal organoids and CRC.

Schematic representation of the discovery of colibactin-induced mutational signatures using organoid exposure and detection of these signatures in CRC whole-genome sequencing data.

These findings open interesting questions for the implementation of early detection and prevention strategies in the future. It will be important to determine the prevalence of pks⁺ E. coli across different population groups, age segments and linking it to CRC development. Intriguingly, SBS88 and ID18 have been found in a subset of non-cancerous colorectal crypts of healthy donors⁹ and in patients with inflammatory bowel disease.¹⁰ The shared ancestral mutations between colonic crypts indicate that the mutations were most likely induced within the first decade of life. The colibactin signatures are among the very few mutagenic processes in the colon with an identified and potentially preventable cause. We therefore envision that future preventive strategies may encompass targeted depletion of genotoxic bacterial species, interference with the mutagenic action of colibactin or its production by pks⁺ bacteria. Such interventions may be informed by recent key advances regarding the structure of colibactin^6,7 and its interaction with specific DNA sequences in the process of double strand break induction.¹¹

Besides the colon, several other tissues display the mutational patterns linked to pks⁺ E. coli. Interestingly, some cases of head and neck and urinary tract cancer in our cohort had a strikingly high contribution of SBS88/ID18 to their mutation load. This finding is in line with the discovery of an oral squamous cell carcinoma¹² containing similarly high levels of SBS88 and ID18 mutations. The large number of colibactin-induced mutations within these tumours points towards a causal role of infections with pks⁺ E. coli or similar bacteria in some cases of head and neck as well as urinary tract cancers. More detailed studies on the prevalence of colibactin-producing bacteria and SBS88/ID18 in these tissues and cancers will help elucidate the extent and cause of bacterial contributions to these cancers.

To obtain a comprehensive picture of microbially induced mutagenesis, several other bacterial strains and species associated with diverse cancers and their precursors should be investigated. Future efforts may focus on strains of Fusobacterium nucleatum, Bacteroides fragilis, Campylobacter jejuni, Shigella flexneria, Salmonella enterica, Chlamydia trachomatis or Helicobacter pylori which have been shown to exert genotoxicity. As another example, the probiotic strain E. coli Nissle 1917 which harbours the pks island and is used in indications such as inflammatory bowel disease is currently being investigated for its ability to induce the characteristic SBS88/ID18 mutations.

Finally, colibactin induces DNA damage which in turn results in multiple mutation types, such as single base substitutions and small indels. These are likely to arise due to the action of diverse DNA damage response and repair processes. It seems reasonable to think that other genotoxins may leave distinct mutational patterns. Analysis of the genomic alterations in cancer genomes beyond single bases substitutions and short indels¹³ may help to understand the mutagenic effects of microbes in greater detail and uncover new mutagenic processes. This would make it possible to elucidate the full impact of the microbiome on the induction of driver mutations. While our study serves as a proof-of-principle that bacteria can leave specific mutational patterns in cancer genomes, we anticipate that other microbe-induced DNA patterns will be uncovered, each with a unique role in carcinogenesis.

Author contributions

A.R.H., C.P.-M. and J.P. contributed equally to the conceptualisation, writing and editing of the manuscript. C.P.-M. generated the image illustration.

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Data availability

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Competing interests

The authors declare no competing interests.

Funding information

A.R.H., C.P.-M. and J.P. were supported by the Oncode Institute (partly financed by the Dutch Cancer Society). A.R.H. was supported by a VIDI grant from the NWO (no. 016.Vidi.171.023); C.P.-M. and J.P. were supported by the following grants: CRUK OPTIMISTICC (C10674/A27140), the Gravitation project CancerGenomiCs.nl, and the Netherlands Organ-on-Chip Initiative (024.003.001) from the Netherlands Organisation for Scientific Research (NWO) funded by the Ministry of Education, Culture and Science of the government of the Netherlands.