The effects of enteropathogenic Escherichia coli on the classical genetic pathways of colorectal cancer, using in vitro and ex vivo human models

Despite high prevalence and mortality, and an excellent knowledge of the aetiologic genetic changes of sporadic colorectal cancer, the causes of this disease are not well defined. DNA mismatch repair and Wnt signalling (via (3-catenin) are classic genetic pathways altered during colorectal carcinogenesis, currently there is little evidence to suggest how gene-environment interactions could influence these pathways. Recent studies have found that adherent Escherichia coli are associated with colonic adenocarcinomas, leading to speculation that in similarity to gastric cancer, bacterial infection has a central role in colonic tumourigenesis. The attaching and effacing (AE) bacterium enteropathogenic E. coli (EPEC) intimately attaches to the intestinal epithelium and is found in 2.5-10% of healthy children and adults in developed countries. When attaching to host cells, EPEC secretes effector proteins that have wide ranging effects on host molecular biology. The aim of this study was to test the hypothesis that EPEC infection causes molecular changes in host epithelial cells that predispose to neoplastic transformation. Model systems for EPEC infection were successfully established using in vitro co-culture with human colorectal cancer cell lines and co-culture with ex vivo human colonic mucosa; human adenocarcinomas were also probed for the presence of AE E. coli. Immunofluorescence identified mucosa associated AE E. coli in 5/20 (25%) adenocarcinomas. When co-cultured with normal human colonic mucosa, EPEC entered 10.6% of crypts, and closely associated with cells in the proliferative progenitor compartment. Mass spectrometry and microarray analysis validated the in vitro model and revealed a range of proteomic and transcriptomic effects in EPEC infected cells. Western blots and quantitative immunofluorescence demonstrated that EPEC downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 and the Wnt signalling / adhesion protein (3-catenin in vitro. Disruption of DNA mismatch repair is a causative factor in the development of many hereditary and sporadic colorectal cancers, and disruption of cell-cell adhesion has the potential to subvert normal colonic crypt homeostasis. These novel findings therefore suggest that chronic EPEC infection can predispose to cancer development by increasing the susceptibility of colonic epithelial cells to mutation by dietary or other carcinogens, and by altering expression of cytoskeletal and cell attachment protein

An Escherichia coli effector protein promotes host mutation via depletion of DNA mismatch repair proteins.

Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) human pathogen that causes diarrhea during acute infection, and it can also sustain asymptomatic colonization. A/E E. coli depletes host cell DNA mismatch repair (MMR) proteins in colonic cell lines and has been detected in colorectal cancer (CRC) patients. However, until now, a direct link between infection and host mutagenesis has not been fully demonstrated. Here we show that the EPEC-secreted effector protein EspF is critical for complete EPEC-induced depletion of MMR proteins. The mechanism of EspF activity on MMR protein was posttranscriptional and dependent on EspF mitochondrial targeting. EPEC infection also induced EspF-independent elevation of host reactive oxygen species levels. Moreover, EPEC infection significantly increased spontaneous mutation frequency in host cells, and this effect was dependent on mitochondrially targeted EspF. Taken together, these results support the hypothesis that A/E E. coli can promote colorectal carcinogenesis in humans

Immune regulated IDO1-dependent tryptophan metabolism is source of one-carbon units for pancreatic cancer and stellate cells

Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis