Bacteroides fragilis toxin induces epithelial-to-mesenchymal transition and stem-like phenotype in breast epithelial cells and concomitantly activates Notch and βcatenin axes. Bacteroides fragilis toxin induces epithelial-to-mesenchymal transition and stem-like phenotype in breast epithelial cells and concomitantly activates Notch and βcatenin axes

Aided by deep sequencing techniques, recent studies suggest 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. Present study examines the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression. Extensive data mining and metagenomic analyses of existing datasets revealed the presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is a pro-carcinogenic bacterium known for its potential to initiate and promote colon cancer; its pathogenicity has been attributed to its unique toxin ‘BFT’. About 35% of human population asymptomatically harbor ETBF colonization in the gut. Mice with enteric B fragilis infection exhibited a significant amount of circulating BFT and distinct morphological alterations in mammary gland similar to focal hyperplasia. Histological analysis revealed inflammation, fibrosis, breast duct thickening and hyperproliferation of breast epithelial cells. In vitro, upon treatment with BFT, prominent cytoskeletal reorganization, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed 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 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. Mechanistic studies showed that BFT induced expression and nuclear translocation of NICD and β-catenin resulting in activation of downstream targets. Inhibition of Notch1 and β-catenin using γ-secretase and β-catenin inhibitors successfully inhibited functional effects of BFT. Mammary gland implantation and in vivo limiting dilution assays were utilized to corroborate the in vitro findings. BFT-pretreated MCF7 cells exhibit increased tumor growth and form 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. Altered expression of stemness markers was also confirmed by RNA sequencing. Overall design: MCF7 cells were seeded in 10% serum supplemented DMEM media and allowed to grow for 24 hours. Cells were then treated with 100 ng/ml of BFT in 2% serum supplemented DMEM. Medium in the control group was changed to 2% serum supplemented DMEM. 3 days post tretment, cells were harvested in 0.25% trypsin/EDTA, neutralized with 10% FBS supplemented DMEM and centrifuged. Pellets were resuspended in serum free media and equal volume of matrigel was added to the cell suspension. 5 million cells were implanted on to the 4th mammary fat pad of SCID-NOD mice on either side with E2 supplementation. Tumors were allowed to grow for 8 weeks and monitored after which tumors were resected. The resecetd tumors were dissociated and tumor cells were isolated and subjected to in vivo limiting dilution; cells ranging from 5000 to 500000 were implanted in different mammary fat pads of SCID-NOD mice and tumors were allowed to grow. At the end of 8 weeks, resulting tumors were resected and RNA sequencing was performed. RNA sequencing was also performed on parental cells (MCF7 cells treated with 100ng/ml of BFT in vitro as described earlier). There were 16 samples in total, comprising biological replicates for 4 sample classes, including untreated controls.