Abstract PR06: Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes

Background: The last decade established significant contributions of microbiome to many organ-specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast; however, their functional impact and underlying mechanisms are unknown. The present study was designed to examine the contribution of procarcinogenic bacteria in breast cancer initiation, growth, and progression. Results: Utilizing extensive data mining and metagenomic analyses, we discovered the presence of toxin-producing Bacteriode fragilis (B. fragilis) in malignant breast. B. fragilis is a procarcinogenic bacteria known for its potential to initiate and/or promote colon cancer, and its pathogenicity has been attributed to its unique toxin “Fragilysin” or “B. fragilis toxin (BFT).” Mice infected with B. fragilis exhibited a significant increase in circulating BFT and distinct morphologic alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker E-cadherin along with elevated levels of mesenchymal markers, N-cadherin, and vimentin were observed. BFT also increased the expression and nuclear translocation of EMT-related transcription factors, Snail, Slug, and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog, and Sox2). Mechanistic studies showed that BFT induced expression and nuclear translocation of NICD (cleaved NOTCH) and βcatenin, resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, we found that BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior, confirming that brief BFT exposure elicits long-term molecular changes. Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriode fragilis toxin induces EMT, invasion/migration, and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes. This abstract is also being presented as Poster A24. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia Sears, Dipali Sharma. Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR06.