The colonic microbiota is suspected of playing an important role in the etiology of sporadic colorectal cancer (CRC). However, no studies have convincingly defined how bacteria might promote this common tumor. In this grant we link the oxidative physiology of this common intestinal commensal, Enterococcus faecalis, to the origin of chromosomal instability (CIN). Among intestinal commensals E. faecalis is unique in generating extracellular superoxide, promoting oxidative stress, and damaging DNA. We propose a novel hypothesis for sporadic CRC that involves innate immune responses triggered by redox-active commensal bacteria to produce bystander effects (BSE) that lead to CIN in crypt stem cells. The bystander effect refers to collateral DNA damage in neighboring cells from activated cells that produce clastogens (or chromosome breaking factors). The role of lipid aldehydes and their metabolism on the susceptibility of epithelial target cells to CIN will be evaluated. The ability of E. faecalis to induce cyclooxygenase-2, prostanoids, TNF1, malondialdehyde and 4- hydroxynonenal in the colonic mucosa of interleukin (IL)-10 knockout mice will be determined. The role of macrophages in the production of inflammation, dysplasia, and tumor formation in E. faecalis colonized mice will be evaluated. The effect of buthionine sulfoximine (BSO) and N-acetyl-L-cysteine (NAC) as inhibitors or promoters, respectively, of glutathione and glutathione S-transferases will be tested. The generation of mutagenic exocyclic DNA adducts and DNA double strand breaks (DSBs) in target cells by the macrophage-induced BSE will be measured using LC/MS/MS, 32P-postlabeling techniques, and immunohistochemistry for 3H2AX. Mutations in the colon due to E. faecalis colonization will be quantified using a hybrid IL-10-/-/Big Blue. Mutant model. Finally, the ability of macrophage-induced BSE to transform a primary epithelial cell line will be performed using a nude mouse xenograft model. Genetic and epigenetic changes typically associated with sporadic CRC will be assessed in transformed cells. The goal of this grant is to elucidate potential mechanisms of colonic carcinogenesis that involve commensals. Colonization of IL-10-/- mice by E. faecalis will be used to test NAC, a glutathione precursor, as a potential chemopreventive. In this fashion we hope to define mechanistic and molecular processes involved in sporadic CRC carcinogenesis and identify agents to novel targets for chemoprevention. Sporadic colorectal cancer is a leading cause of cancer and cancer death worldwide. These tumors are believed to arise, in part, because of bacteria in the colon. In this project the unusual oxidative nature of a common intestinal bacterium, Enterococcus faecalis, will be tested for its ability to damage DNA in cells lining the colon and thereby promote the development of mutations that lead to cancer. These results will identify new approaches and targets for preventing sporadic colorectal cancer. PUBLIC HEALTH RELEVANCE Sporadic colorectal cancer is a leading cause of cancer and cancer death worldwide. These tumors are believed to arise, in part, because of bacteria in the colon. In this project the unusual oxidative nature of a common intestinal bacterium, Enterococcus faecalis, will be tested for its ability to damage DNA in cells lining the colon and thereby promote the development of mutations that lead to cancer. These results will identify new approaches and targets for preventing sporadic colorectal cancer.

R01CA127893

2008-07-01

2014-05-31