Project description:Colorectal cancer is driven by a sequential cascade of mutations known as the adenoma-carcinoma sequence. Recent studies have revealed that specific bacterial species present in the colonic microbiota can induce mutations and contribute to this malignancy. Specifically, genotoxic colibactin-producing pks+ Escherichia coli strains can induce DNA double strand breaks (DSBs) and promote tumor development in mouse models of colorectal cancer. Here, we investigated the transformation potential of colibactin by using organoids and polarized monolayers derived from primary murine colon epithelial cells and reveal striking phenotypic changes upon short-term infection. This study demonstrates the direct pro-oncogenic potential of pks+ E. coli, as such transformations in vivo could facilitate colitis-associated colorectal carcinogenesis.
Project description:Colibactin, a potent genotoxin of Escherichia coli, causes DNA double strand breaks (DSBs). We investigated if colibactin creates a particular DNA damage signature in infected human cells. Genomic contexts of colibactin-induced DSBs were enriched for a distinct AT-rich hexameric sequence motif. A survey of somatic mutations at the colibactin target sites of several thousand cancer genomes revealed significant enrichment of the motif in colorectal cancers. Moreover, the exact break point location corresponded with mutational hot spots in these cancers corresponding to a distinct trinucleotide signature. This work provides evidence for a role of colibactin in the etiology of human cancer.
Project description:The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic used for chronic multidrug resistant infections infections, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can augment colorectal cancer (CRC) through DNA double stranded breaks and interstrand crosslinks. While the structure and biosynthesis of colibactin has been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by pathogenic E. coli are largely unexplored. This research highlights the regulation of the colibactin-producing biosynthetic gene cluster under polymyxin stress. Using a multi-omic approach, we have identified that polymyxin stress enhances the abundance of colibactin biosynthesis proteins (Clb’s) in multiple pks+ E. coli strains, including pro-carcinogenic AIEC: NC101, the probiotic strain: E. coli Nissle 1917, and the antibiotic testing strain: E. coli ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb’s by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E. coli with heightened tolerance for polymyxin antibiotics induced greater DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E. coli. Ultimately, our findings will inform future studies investigating colibactin regulation, the microbial response to antibiotics in the gut, and the ability of seemingly innocuous commensal microbes to induce host disease.
Project description:By comparing the transcriptome profile upon deletion or overexpression of ClbR or ClbQ, we addressed the role of these proteins in regulation of colibactin expression. Our global screen indicates that ClbR is the key transcriptional activator of colibactin expression. In addition, we further investigated potential transcription start sites of the colibactin determinant.
Project description:Colon cancer is initiated by stem cells that escape the strict control. In most colon tumors this process is driven through aberrant activation of Wnt signaling by mutations that occur in components acting downstream of the receptor complex and that unfetter tumor cells from the need for Wnt ligands. Here we describe a special type of colon cancer that does not depend on mutated core components of the Wnt pathway. Genetically blocking Wnt secretion from epithelial cells of such tumors results in apoptosis, reduced expression of colon cancer markers, followed by enhanced tumor differentiation. In contrast to the normal colonic epithelium, such tumor cells auto-secrete Wnt ligands to maintain their uncontrolled proliferative behavior. In humans, we determined certain cases of colon cancers in which the Wnt pathway is hyperactive, but not through mutations in its core components. Our findings illuminate the path in therapy to find further subtypes of Wnt- dependent colon cancer, that might be responsive to Wnt secretion inhibitors.
Project description:Colibactin, a bacterial genotoxin produced by E. coli harboring the pks genomic island, induces cytopathic effects such as DNA breaks, cell cycle arrest and apoptosis. Patients with a colonic dysfunction due to inflammatory bowel disease such as ulcerative colitis have an elevated likelihood of carrying pks+ E. coli in their colon microbiota but it is not clear whether and how they contribute to the pathogenesis of colitis. Using a gnotobiotic mouse model, we show that pks+ E. coli do not affect colonic integrity under homeostatic conditions, with the microbiota remaining separated from the epithelium by a mucus barrier. However, upon chemical disruption of this barrier by DSS, the microbes gain direct access to the epithelium, causing severe epithelial injury, and development of colitis, while mice colonized with an isogenic ΔclbR mutant incapable of producing colibactin suffer significantly less pronounced effects. While ΔclbR-colonized animals show efficient recovery of the mucus barrier and crypt homeostasis, recovery in WT-colonized mice is impaired. Instead, the mucosa remains in a chronic regenerative state characterized by high proliferation and impaired differentiation of enterocytes and goblet cells, preventing the re-establishment of a functional barrier. In turn, pks+ E. coli remain in direct contact with the epithelium, perpetuating the process and triggering chronic mucosal inflammation that morphologically and transcriptionally resembles human ulcerative colitis. It is characterized by high levels of stromal R-spondin 3. Genetic overexpression of R-spondin 3 in colon myofibroblasts is sufficient to mimic this chronic regenerative state, resulting in barrier disruption and expansion of E. coli. Together, our data reveal that pks+ E. coli are pathobionts that upon contact with the epithelium promote severe injury and interfere with recovery, initiating chronic tissue dysfunction and inflammation.
Project description:Interventions: Colibactin positive patients are given biolactis (3 g/day) for 3 months.
Primary outcome(s): Detection of colibactin-producing bacteria 90 days after the start of the intervention.
Study Design: single arm study, open(masking not used), no treatment control/standard of care control, single assignment, basic science
Project description:Experimental set accompanying Giacomini et al publication "A legacy gene-expression signature of genetic instability in colon cancer". Includes 18 colon cancer cell line training set, 13 colon cancer cell line test set, and 3 cell lines (HCT116, HCT116+ch2, HCT116+ch3) used to evaluate signature after correcting underlying genetic instability. Experiments were performed by comparing mRNA from each colon cancer cell line (Cy5; channel 2) to a "universal" mRNA reference (Cy3; channel 1). A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. Series type: disease_state_design Series_overall_design: Using regression correlation