Infection with genotoxin-producing Salmonella enterica synergises with loss of the tumour suppressor APC in promoting genomic instability via the PI3K pathway in colonic epithelial cells.

Océane C.B Martin,Teresa Frisan,Maria G Masucci,Jerry W Shay,Jacques Dupuy,Puran Chen,Anna Bergonzini,Federica D'Amico,Mattias Svensson

doi:10.1111/cmi.13099

Abstract

Several commensal and pathogenic Gram‐negative bacteria produce DNA‐damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin‐producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin‐producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC‐deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3‐kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin‐producing bacteria in promoting a microenvironment conducive to malignant transformation.

Highlights

Colorectal cancer (CRC) is the third most common cancer (IARC, n.d.) and a leading cause of cancer death
We have assessed the consequences of infection with Salmonella enterica, the only genotoxin‐producing bacterium associated with an increased risk of developing hepatobiliary and colon carcinoma in humans (Dutta, Garg, Kumar, & Tandon, 2000; Mughini‐ Gras et al, 2018) on alteration of the DNA damage response (DDR) and acquisition of genomic instability in normal and adenomatous polyposis coli (APC)‐deficient cells, using classical two‐dimensional (2D) and organotypic three‐dimensional (3D) tissue models
Two cell models were used to investigate whether APC deficiency alters the outcome of infection with genotoxin‐producing bacteria in eukaryotic cells: (a) the immortalized non transformed human colonic epithelial cell line 1CT that carries a functional APC gene (Roig et al, 2010) and the isogenic 1CTA cell line where a three‐fold downregulation of the APC mRNA was obtained by stable transfection of specific shRNA (Graillot et al, 2016; Figure 1a); and (b) the SV40 large T antigen immortalized murine colonic epithelial cells that are either APC wild type (Apc+/+) or APC haploinsufficient due to an allelic mutation leading to expression of a truncated inactive product (Apc+/Min; Forest, Clement, Pierre, Meflah, & Menanteau, 2003)

Summary

| INTRODUCTION

Colorectal cancer (CRC) is the third most common cancer (IARC, n.d.) and a leading cause of cancer death (reviewed in Siegel, Desantis, & Jemal, 2014). The introduction of generation sequencing has highlighted significant alterations in the intestinal microbiota of CRC patients compared with healthy subjects (Villeger et al, 2018) It is still unknown whether the dysbiosis precedes or is a consequence of the tumour microenvironment and whether microbial products contribute to the carcinogenic process. The genotoxic activity of these bacterial effectors may contribute to CRC development To address this issue, we have assessed the consequences of infection with Salmonella enterica, the only genotoxin‐producing bacterium associated with an increased risk of developing hepatobiliary and colon carcinoma in humans (Dutta, Garg, Kumar, & Tandon, 2000; Mughini‐ Gras et al, 2018) on alteration of the DDR and acquisition of genomic instability in normal and APC‐deficient cells, using classical two‐dimensional (2D) and organotypic three‐dimensional (3D) tissue models. We found that infection with genotoxin‐producing Salmonella synergises with the loss of APC to enhance genomic instability both in 2D and

| RESULTS

| DISCUSSION

| EXPERIMENTAL PROCEDURES

Findings

CONFLICT OF INTEREST