B-351 Genotoxic Producing Escherichia coli Protects and Promotes Breast Adenocarcinoma

Abstract

Abstract Background Recent studies have linked certain colibactin producing subphylas of E. coli to colorectal cancer. The bacterium synthesizes colibactin via the pks genomic island containing a series of clbA to clbS genes. The genotoxin causes inter-strand DNA cross-links, which lead to double strand breaks and promotion of colorectal adenocarcinoma. The published studies have prioritized the colorectal regions of the body where E. coli is commonly found, but none have identified the same genotoxic relationship with other types of adenocarcinoma. In this study, we evaluate DNA damage and cellular changes of mammary breast tissue based on high and low levels of colibactin producing E. coli exposure. The goal is to evaluate if colibactin can affect cells in the mammary breast tissue promoting breast adenocarcinoma growth. Methods Two cell lines were acquired from ATCC: primary mammary epithelial cells (PCS-600-010) and MCF-7 breast adenocarcinoma cells (HTB-22). Both cell lines were split into six culture flasks and maintained until ∼80% confluence was reached using the ATCC cell culture protocols. Once all the flasks had reached the target confluence, they were divided into exposure groups of high and low concentrations of colibactin producing E. coli, a group of non-colibactin producing E. coli, a group exposed only to phosphate buffered saline, and a control group with no exposure. Cellular morphology was observed using light microscopy before, after exposure, and every 5 minutes up to 15 minutes. E. coli and colibactin DNA concentrations were quantified using quantitative polymerase chain reaction. Genomic DNA was extracted from each group of cells. The DNA from each group was whole genome sequenced and analyzed for genomic variation. Results The normal cells exposed to E. coli showed immediate lysing and continued to lyse after a 5 minute incubation; however, the adenocarcinoma positive cells showed no lysing up to 30 minutes incubation. Sequencing results show genomic variation between the groups and specific break points associated with the E. coli exposed groups. Conclusions Our results show that the presence of E. coli with mammary breast tissue can destroy normal tissue, promote lysis resistance, and allow adenocarcinoma cells to continue to grow. The colibactin producing E. coli does cause double stranded breaks and can lead to cell death. This new information points out a critical need to monitor the microbiome and infection levels of people at risk or in early stages of cancer to prevent adenocarcinoma growth and destruction of normal tissue. This study indicates that colibactin producing E. coli may play a larger role in tissue damage and promotion of other types of cancer. More research is needed to elucidate the molecular pathway and mechanisms of colibactin, which will lead to new diagnostics or therapeutics that monitor E. coli levels or inhibit colibactin’s toxic effects.