Bacteroides fragilis toxin induces epithelial-to-mesenchymal transition and stem-like phenotype in breast epithelial cells and concomitantly activates Notch and ßcatenin 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 ß-catenin resulting in activation of downstream targets. Inhibition of Notch1 and ß-catenin using ?-secretase and ß-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.

借助深度测序（deep sequencing）技术，近期研究表明，与正常乳腺组织相比，病变乳腺中存在独特的乳腺微生物群（breast microbiota），且微生物群落组成发生显著改变。然而，其功能影响与潜在作用机制仍未明确。
本研究旨在探究促癌细菌在乳腺癌发生、发展及进程中的作用。通过对现有数据集进行广泛的数据挖掘与宏基因组分析（metagenomic analyses），本研究在恶性乳腺组织中检出了产毒素的脆弱拟杆菌（Bacteroides fragilis）。脆弱拟杆菌是一种已知可诱发并促进结肠癌发生的促癌细菌，其致病性与其独特毒素‘BFT’密切相关。约35%的人群在肠道中无症状定植产肠毒素脆弱拟杆菌（Enterotoxigenic Bacteroides fragilis, ETBF）。
肠道感染脆弱拟杆菌的小鼠可检测到显著水平的循环BFT，且乳腺组织出现与局灶性增生相似的明显形态学改变。组织学分析显示，小鼠乳腺出现炎症、纤维化、乳腺导管增厚以及乳腺上皮细胞过度增殖。
体外实验中，经BFT处理的MCF10A与MCF7细胞出现显著的细胞骨架重排，迁移与侵袭能力显著增强，黏附能力下降，同时检测到上皮间质转化（epithelial-to-mesenchymal transition, EMT）相关分子标志物的表达变化：上皮标志物E-钙黏蛋白（E-cadherin）表达下调，而间质标志物N-钙黏蛋白（N-cadherin）与波形蛋白（vimentin）表达水平上调。此外，BFT还可上调EMT相关转录因子Snail、Slug及Twist的表达。经BFT处理的细胞获得了干细胞样表型，其形成二级与三级乳腺球的能力显著增强。
机制研究表明，BFT可诱导Notch胞内结构域（Notch Intracellular Domain, NICD）与β-连环蛋白（β-catenin）的表达及核转位，从而激活下游靶基因。使用γ-分泌酶（γ-secretase）抑制剂与β-连环蛋白抑制剂分别抑制Notch1与β-连环蛋白的活性，可有效阻断BFT介导的生物学功能。
本研究通过乳腺植入实验与体内极限稀释实验验证了体外实验结果。经BFT预处理的MCF7细胞在小鼠体内的肿瘤生长速度更快，且可形成多灶性肿瘤。使用经BFT预处理的MCF7细胞来源的乳腺肿瘤进行体内极限稀释实验，结果显示肿瘤起始细胞数量显著增加。对这些肿瘤的后续分析显示，其迁移、侵袭及乳腺球形成能力均显著增强，证实短暂的BFT暴露可引发长期的分子层面改变。RNA测序（RNA sequencing）也证实了干细胞标志物的表达发生了改变。

实验整体设计如下：将MCF7细胞接种于添加10%胎牛血清的DMEM培养基中，培养24小时。随后，将细胞置于添加2%胎牛血清的DMEM培养基中，加入终浓度为100 ng/ml的BFT进行处理；对照组细胞则更换为仅添加2%胎牛血清的DMEM培养基。处理3天后，使用0.25%胰蛋白酶-EDTA消化收集细胞，用添加10%胎牛血清的DMEM培养基中和胰蛋白酶活性，随后离心收集细胞沉淀。将细胞沉淀重悬于无血清培养基中，并加入等体积的基质胶（Matrigel）。将5×10^6个细胞接种于SCID-NOD小鼠（SCID-NOD mice）双侧第4乳腺脂肪垫，同时给予雌激素（E2）补充处理。让肿瘤生长8周并定期监测，随后切除肿瘤。将切除的肿瘤组织解离并分离肿瘤细胞，用于体内极限稀释实验：将5000至500000个不等的肿瘤细胞接种于SCID-NOD小鼠的不同乳腺脂肪垫中，继续培养至肿瘤形成。8周后，切除形成的肿瘤并进行RNA测序；同时对亲本细胞（即前文所述经100 ng/ml BFT体外处理的MCF7细胞）进行RNA测序。本实验共包含16个样本，涵盖4组样本类别的生物学重复，其中包括未处理的对照组。