colibactin and Colonic-Neoplasms

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

Topics: Animals; Colonic Neoplasms; Colorectal Neoplasms; DNA Mismatch Repair; Escherichia coli; Humans; Mice; Mutagens; Mutation

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.

Topics: Animals; Chromosome Aberrations; Colon; Colonic Neoplasms; Colorectal Neoplasms; DNA Damage; Epithelial Cells; Escherichia coli; Genomics; Male; Mice; Mice, Knockout; Mutation; Organoids; Peptides; Polyketides

Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101.. Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis.. Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells.. Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.

Topics: Caco-2 Cells; Carcinogenesis; Cellular Senescence; Colonic Neoplasms; DNA Damage; Escherichia coli; Genomic Islands; Humans; Mutagens; Oligosaccharides; Peptides; Polyketides

Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APC

Topics: Animals; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Drug Resistance, Neoplasm; Escherichia coli; Female; Humans; Immunotherapy; Lymphocyte Count; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred C57BL; Neutrophils; Peptides; Polyketides; Programmed Cell Death 1 Receptor; Tumor Microenvironment

Colon cancer is one of the most common malignancies and the fourth leading cause of cancer-related mortality in the world. Colibactin, which is synthesized by the pks genomic island of E. coli interfere with the eukaryotic cell cycle. Cinnamon has an antimicrobial effect and considered as a colon cancer-preventing agent. The aim of the study was to evaluate the effects of cinnamon extract and cinnamaldehyde on clbB gene expression and biofilm formation in clinical isolates of E. coli.. Thirty E. coli carrying pks gene were isolated from the colon cancer patients, inflammatory bowel disease and healthy subjects. Antibiotic susceptibility was evaluated by disk diffusion method and the minimum inhibitory concentration of cinnamon essential oil and cinnamaldehyde by microdilution broth method. In vitro biofilm formation of E.coli isolates was monitored using a microtiter plate method. The presence of clbB, clbA and clbQ genes in E.coli isolates were evaluated by PCR. The effect of cinnamaldehyde and cinnamon essential oil on clbB gene expression was evaluated by Real-Time PCR.. The highest antibiotic resistance was obtained with 94.4% for ticarcillin-clavulanic acid, azithromycin, amoxicillin, and amikacin. The MIC for all clinical isolates was 32 μl/ml of cinnamon essential oil and the MIC of cinnamaldehyde was between 0.00002 to 0.03 μl/ml. After exposure of isolates to cinnamon extract and cinnamaldehyde, 40 and 13.3% were weakly biofilm producers, respectively. The frequencies of clbB, clbA, and clbQ genes were 23.3, 23.3, and 26.7%, respectively. The expression of clbB gene in the presence of the Sub-MIC concentration of cinnamon essential oil and cinnamaldehyde was decreased in 8 isolates compared to untreated isolates (p-value < 0.05).. The antibacterial activity of cinnamaldehyde and cinnamon essential oil allows the use of these herbal compounds for treatment or supplements in infections caused by E. coli and in patients with suspected colorectal cancer.

Topics: Anti-Infective Agents; Biofilms; Cinnamomum zeylanicum; Colonic Neoplasms; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Bacterial; Humans; Oils, Volatile; Peptides; Phytotherapy; Plant Extracts; Polyketides

Topics: Carcinogenesis; Colonic Neoplasms; DNA Damage; Escherichia coli; Escherichia coli Proteins; HeLa Cells; Humans; Mutagens; Peptides; Phosphotransferases (Phosphate Group Acceptor); Polyketides; Virulence

Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of

Topics: Adenomatous Polyposis Coli; Animals; Bacterial Toxins; Bacteroides fragilis; Biofilms; Carcinogenesis; Colon; Colonic Neoplasms; DNA Damage; Escherichia coli; Gastrointestinal Microbiome; Humans; Interleukin-17; Intestinal Mucosa; Metalloendopeptidases; Mice; Peptides; Polyketides; Precancerous Conditions

It is now widely recognized that a range of human diseases, including obesity, cancer and inflammatory bowel disease, is strongly linked to the microbiota. For decades, the microbiota has been proposed to contribute to the pathogenesis of colon cancer. Our recent work reveals that the organization of the mucosal microbiota into biofilms marks a subset of human colon cancer. Further, biofilm-positive colon mucosa in the colon cancer host yields an infrequently detected polyamine metabolite, N(1), N(12)-diacetylspermine, that deserves further study to determine its utility as a marker for colon neoplasia.

Topics: Bacterial Toxins; Bacteroides fragilis; Biofilms; Colonic Neoplasms; Escherichia coli; Fusobacterium nucleatum; Gastrointestinal Microbiome; Humans; Intestinal Mucosa; Metalloendopeptidases; Peptides; Polyketides; Spermine

Intestinal dysbiosis has been reported in patients with colorectal cancer, and there is a high prevalence of Escherichia coli belonging to B2 phylogroup and producing a genotoxin, termed colibactin. Macrophages are one of the predominant tumor-infiltrating immune cells supporting key processes in tumor progression by producing protumoral factors such as cyclooxygenase-2 (COX-2). Here, we investigated whether B2 E. coli colonizing colon tumors could influence protumoral activities of macrophages. In contrast to commensal or nonpathogenic E. coli strains that were efficiently and rapidly degraded by macrophages at 24 h after infection, colon cancer-associated E. coli were able to resist killing by human THP-1 macrophages, to replicate intracellularly, and to persist inside host cells until at least 72 h after infection. Significant increases in COX-2 expression were observed in macrophages infected with colon cancer E. coli compared with macrophages infected with commensal and nonpathogenic E. coli strains or uninfected cells at 72 h after infection. Induction of COX-2 expression required live bacteria and was not due to colibactin production, as similar COX-2 levels were observed in macrophages infected with the wild-type colon cancer-associated E. coli 11G5 strain or a clbQ mutant unable to produce colibactin. Treatment of macrophages with ofloxacin, an antibiotic with intracellular tropism, efficiently decreased the number of intracellular bacteria and suppressed bacteria-induced COX-2 expression. This study provides new insights into the understanding of how tumor- infiltrating bacteria could influence cancer progression through their interaction with immune cells. Manipulation of microbes associated with tumors could have a deep influence on the secretion of protumoral molecules by infiltrating macrophages.

Topics: Cell Line, Tumor; Colonic Neoplasms; Cyclooxygenase 2; Dinoprostone; Escherichia coli; Host-Pathogen Interactions; Humans; Immunoblotting; Macrophages; Microbial Viability; Microscopy, Confocal; Microscopy, Electron, Transmission; p38 Mitogen-Activated Protein Kinases; Peptides; Polyketides; Vacuoles

Escherichia coli strains harbouring the pks island (pks+ E. coli) are often seen in human colorectal tumours and have a carcinogenic effect independent of inflammation in an AOM/IL-10(-/-) (azoxymethane/interleukin) mouse model.. To investigate the mechanism sustaining pks+ E. coli-induced carcinogenesis.. Underlying cell processes were investigated in vitro and in vivo (xenograft model) using intestinal epithelial cells infected by pks+ E. coli or by an isogenic mutant defective for pks (pks- E. coli). The results were supported by data obtained from an AOM/DSS (azoxymethane/dextran sodium sulphate) colon cancer mouse model and from human colon cancer biopsy specimens colonised by pks+ E. coli or pks- E. coli.. Colibactin-producing E. coli enhanced tumour growth in both xenograft and AOM/DSS models. Growth was sustained by cellular senescence (a direct consequence of small ubiquitin-like modifier (SUMO)-conjugated p53 accumulation), which was accompanied by the production of hepatocyte growth factor (HGF). The underlying mechanisms involve microRNA-20a-5p, which targets SENP1, a key protein regulating p53 deSUMOylation. These results are consistent with the expression of SENP1, microRNA-20a-5p, HGF and phosphorylation of HGF receptor found in human and mouse colon cancers colonised by pks+ E. coli.. These data reveal a new paradigm for carcinogenesis, in which colibactin-induced senescence has an important role.

Topics: Animals; Carcinogenesis; Cellular Senescence; Colonic Neoplasms; Cysteine Endopeptidases; Endopeptidases; Escherichia coli; Hepatocyte Growth Factor; Humans; Mice; Mutagens; Mutation; Neoplasms, Experimental; Nuclear Proteins; Peptides; Polyketides; Proto-Oncogene Proteins c-met

Topics: Animals; Carcinogenesis; Colonic Neoplasms; Escherichia coli; Humans; Peptides; Polyketides

The gut microbiota is suspected to promote colorectal cancer (CRC). Escherichia coli are more frequently found in CCR biopsies than in healthy mucosa; furthermore, the majority of mucosa-associated E. coli isolated from CCR harbors the pks genomic island (pks+ E. coli) that is responsible for the synthesis of colibactin, a genotoxic compound. We have recently reported that transient contact of a few malignant cells with colibactin-producing E. coli increases tumor growth in a xenograft mouse model. Growth is sustained by cellular senescence that is accompanied by the production of growth factors. We demonstrated that cellular senescence is a consequence of the pks+ E. coli-induced alteration of p53 SUMOylation, an essential post-translational modification in eukaryotic cells. The underlying mechanisms for this process involve the induction of miR-20a-5p expression, which targets SENP1, a key protein in the regulation of the SUMOylation process. These results are consistent with the expression of SENP1, miR-20a-5p and growth factors that are observed in a CRC mouse model and in human CCR biopsies colonized by pks+ E. coli. Overall, the data reveal a new paradigm for carcinogenesis in which pks+ E. coli infection induces cellular senescence characterized by the production of growth factors that promote the proliferation of uninfected cells and, subsequently, tumor growth.

Topics: Animals; Colonic Neoplasms; Cysteine Endopeptidases; Endopeptidases; Escherichia coli; Mice; MicroRNAs; Models, Biological; Mutagens; Peptides; Polyketides; Sumoylation; Tumor Microenvironment; Tumor Suppressor Protein p53

Commensal Escherichia coli residing in the human gut produce colibactin, a small-molecule genotoxin of unknown structure that has been implicated in the development of colon cancer. Colibactin biosynthesis is hypothesized to involve a prodrug resistance strategy that entails initiation of biosynthesis via construction of an N-terminal prodrug scaffold and late-stage cleavage of this structural motif during product export. Here we describe the biochemical characterization of the prodrug synthesis, elongation, and cleavage enzymes from the colibactin biosynthetic pathway. We show that nonribosomal peptide synthetases ClbN and ClbB assemble and process an N-acyl-D-asparagine prodrug scaffold that serves as a substrate for the periplasmic D-amino peptidase ClbP. In addition to affording information about structural features of colibactin, this work reveals the biosynthetic logic underlying the prodrug resistance strategy and suggests that cytotoxicity requires amide bond cleavage.

Topics: Cell Proliferation; Colonic Neoplasms; Drug Resistance, Neoplasm; Escherichia coli; Humans; Molecular Conformation; Peptide Synthases; Peptides; Polyketides; Prodrugs