Enterotoxigenic Bacteroides Fragilis Research Articles

Single-Cell Profiling Reveals the Impact of Genetic Alterations on the Differentiation of Inflammation-Induced Murine Colon Tumors

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (MinApcΔ716/+) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAFV600E mutation (BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+, BLM) or knocking out Msh2 (Msh2LoxP/LoxPVil1-creMinApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.

Abstract 1259: HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration

Abstract Cancer cells undergo epigenetic reprogramming to co-opt stem cell networks and drive tumor progression, although how this occurs remains unclear. The High Mobility Group A1 (HMGA1) gene encodes an architectural transcription factor that binds to DNA at AT-rich sequences where it recruits chromatin complexes to modulate gene expression. HMGA1 is highly expressed during embryogenesis and in adult stem cells, but silenced in differentiated cells. HMGA1 becomes re-expressed in aggressive cancers where high levels portend adverse outcomes. In colorectal cancer (CRC), HMGA1 is among the genes most highly overexpressed compared to normal colon epithelium. We previously discovered that HMGA1 is required for oncogenic phenotypes in CRC cell lines by inducing genes involved in an epithelial-mesenchymal transition. Hmga1 transgenic mice also develop aberrant proliferation and polyposis of small intestinal epithelium where Hmga1 amplifies Wnt signals to enhance self-renewal. To determine how HMGA1 functions in colon epithelial homeostasis and tumorigenesis, we examined mouse models with overexpression or deficiency of Hmga1. Here, we uncover a previously unknown role for HMGA1 as an epigenetic gatekeeper of Wnt signals in colon epithelial cells, both during tumorigenesis driven by mutant APC or stress-induced tissue repair. Strikingly, loss of just a single Hmga1 allele within the colon epithelium disrupts tumorigenesis while prolonging survival in two models of colon tumorigenesis driven by APC mutation, including CDX2P-CreERTCApcfl/fl and APCMin mice inoculated with pro-carcinogenic, enterotoxigenic Bacteroides fragilis. Single cell RNA sequencing in Apc mutant crypt epithelial cells reveals that intact Hmga1 is required for expansion in the Lgr5+ stem cell pool, deep secretory cells, and tuft cells during tumorigenesis. Hmga1 deficiency impacts the stem cell pool the greatest, decreasing Lgr5+ stem cells with skewing to more proliferative, transit amplifying-like cells. Single cell transcriptomic analyses together with assays of single cell chromatin accessibility demonstrate that Hmga1 maintains accessible chromatin to activate gene loci involved in Wnt signals, including Ascl2, a stem cell master regulator, Wnt agonist receptors, and Wnt downstream effectors. Surprisingly, either depletion or overexpression of Hmga1 has no impact on colon epithelial regeneration under homeostatic conditions, although Hmga1 overexpression enhances tissue repair and survival following injury induced by inflammation or irradiation. Together, our results establish HMGA1 as an epigenetic gatekeeper of Wnt signals in colon stem cells during APC-driven tumorigenesis or injury, but not steady state homeostasis, highlighting its role as a promising therapeutic target for both cancer and regenerative medicine. Citation Format: Linda M. S. Resar, Li Z. Luo, Lingling Xian, Iliana Herrera, Shaoguang Wu, Jung-Hyun Kim, Ying Feng, Julian Calderon-Espinosa, Eric R. Fearon, Cynthia Sears. HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1259.

Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1.

Tumor-resident microbiota in breast cancer promote cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increases the chemosensitivity of breast cancer by impairing BCSCs.

Dissecting Gut-Microbial CommunityInteractions using a Gut Microbiome-on-a-Chip.

While the human gut microbiota has a significant impact on gut health and disease, understanding of the roles of gut microbes, interactions, and collective impact of gut microbes on various aspects of human gut health is limitedby the lack of suitable in vitro model system that can accurately replicate gut-like environment and enable the close visualization on causal and mechanistic relationships between microbial constitutents and the gut. , In this study, we present a scalable Gut Microbiome-on-a-Chip (GMoC) with great imaging capability and scalability, providing a physiologically relevantdynamic gut-microbes interfaces. This chip features a reproducible 3D stratified gut epithelium derived from Caco-2 cells(µGut), mimickingkey intestinal architecture, functions, and cellular complexity, providing a physiolocially relevant gut environment for microbes residing in the gut. Incorporating tumorigenic bacteria, enterotoxigenic Bacteroides fragilis (ETBF), into the GMoC enable the observation of pathogenic behaviors of ETBF, leading to µGut disruption and pro-tumorigenic signaling activations. Pre-treating the µGut with a beneficial gut microbe Lactobacillus spp., effectively prevent ETBF-mediated gut pathogenesis, preserving the healthy state of the µGut through competition-mediated colonization resistance. The GMoC holds potential as a valuable tool for exploring unknown roles of gut microbes in microbe-induced pathogenesis and microbe-based therapeutic development.

Keystone pathobionts associated with colorectal cancer promote oncogenic reprograming.

Fusobacterium nucleatum (Fn) and enterotoxigenic Bacteroides fragilis (ETBF) are two pathobionts consistently enriched in the gut microbiomes of patients with colorectal cancer (CRC) compared to healthy counterparts and frequently observed for their direct association within tumors. Although several molecular mechanisms have been identified that directly link these organisms to features of CRC in specific cell types, their specific effects on the epithelium and local immune compartment are not well-understood. To fill this gap, we leveraged single-cell RNA sequencing (scRNA-seq) on wildtype mice and mouse model of CRC. We find that Fn and ETBF exacerbate cancer-like transcriptional phenotypes in transit-amplifying and mature enterocytes in a mouse model of CRC. We also observed increased T cells in the pathobiont-exposed mice, but these pathobiont-specific differences observed in wildtype mice were abrogated in the mouse model of CRC. Although there are similarities in the responses provoked by each organism, we find pathobiont-specific effects in Myc-signaling and fatty acid metabolism. These findings support a role for Fn and ETBF in potentiating tumorigenesis via the induction of a cancer stem cell-like transit-amplifying and enterocyte population and the disruption of CTL cytotoxic function.

Enterotoxigenic Bacteroides fragilis (ETBF) Enhances Colorectal Cancer Cell Proliferation and Metastasis Through HDAC3/miR-139-3p Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) is believed to promote the malignant process of colorectal cancer (CRC), but the underlying molecular mechanism still needs to be revealed. CRC cells (SW480 and HCT-116) were treated with ETBF strain. Cell proliferation, invasion and, migration were evaluated by cell counting kit 8 assay, EdU assay, colony formation assay, transwell assay, and wound healing assay. Protein expression was analyzed by western blot. MicroRNA (miR)-139-3p and histone deacetylase 3 (HDAC3) expression levels in tissues and cells were determined by qRT-PCR. Xenograft tumor model was conducted to evaluate the effect of miR-139-3p on CRC tumor growth. ETBF treatment could promote CRC cell proliferation, invasion and migration. MiR-139-3p expression was decreased by ETBF, and its overexpression reversed the effect of ETBF on CRC cell progression. HDAC3 negatively regulated miR-139-3p expression, and its overexpression facilitated CRC cell behaviors via reducing miR-139-3p expression. Moreover, HDAC3 expression was increased by ETBF, and its knockdown also abolished ETBF-mediated CRC cell progression. Additionally, miR-139-3p overexpression could reduce CRC tumor growth in vivo. ETBF aggravated CRC proliferation and metastasis via the regulation of HDAC3/miR-139-3p axis. The discovery of ETBF/HDAC3/miR-139-3p axis may provide a new direction for CRC treatment.

Intratumoral presence of the genotoxic gut bacteria pks+E. coli, Enterotoxigenic Bacteroides fragilis, and Fusobacterium nucleatum and their association with clinicopathological and molecular features of colorectal cancer

BackgroundThis study aimed to investigate clinicopathological and molecular tumour features associated with intratumoral pks+Escherichia coli (pks+E.coli+), pks+E.coli- (non-E.coli bacteria harbouring the pks island), Enterotoxigenic Bacteroides fragilis (ETBF) and Fusobacterium nucleatum (F. nucleatum).MethodsWe screened 1697 tumour-derived DNA samples from the Australasian Colorectal Cancer Family Registry, Melbourne Collaborative Cohort Study and the ANGELS study using targeted PCR.ResultsPks+E.coli+ was associated with male sex (P < 0.01) and APC:c.835-8 A > G somatic mutation (P = 0.03). The association between pks+E.coli+ and APC:c.835-8 A > G was specific to early-onset CRCs (diagnosed<45years, P = 0.02). The APC:c.835-A > G was not associated with pks+E.coli- (P = 0.36). F. nucleatum was associated with DNA mismatch repair deficiency (MMRd), BRAF:c.1799T>A p.V600E mutation, CpG island methylator phenotype, proximal tumour location, and high levels of tumour infiltrating lymphocytes (Ps < 0.01). In the stratified analysis by MMRd subgroups, F. nucleatum was associated with Lynch syndrome, MLH1 methylated and double MMR somatic mutated MMRd subgroups (Ps < 0.01).ConclusionIntratumoral pks+E.coli+ but not pks+E.coli- are associated with CRCs harbouring the APC:c.835-8 A > G somatic mutation, suggesting that this mutation is specifically related to DNA damage from colibactin-producing E.coli exposures. F. nucleatum was associated with both hereditary and sporadic MMRd subtypes, suggesting the MMRd tumour microenvironment is important for F. nucleatum colonisation irrespective of its cause.

Entero-toxigenic Bacteroides fragilis contributes to intestinal barrier injury and colorectal cancer progression by mediating the BFT/STAT3/ZEB2 pathway

ABSTRACT Our previous findings confirmed the high enrichment of Bacteroides fragilis (BF) in fecal samples from patients with colorectal cancer (CRC). The intestinal mucosal barrier is the first defense of the organism against commensal flora and intestinal pathogens and is closely associated with the occurrence and development of CRC. Therefore, this study aimed to investigate the molecular mechanisms through which BF mediates intestinal barrier injury and CRC progression. SW480 cells and a Caco2 intestinal barrier model were treated with entero-toxigenic BF (ETBF), its enterotoxin (B. fragilis toxin, BFT), and non-toxigenic BF (NTBF). Cell counting kit-8, flow cytometry, wound healing and transwell assays were performed to analyze the proliferation, apoptosis, migration, and invasion of SW480 cells. Transmission electron microscopy, FITC-dextran, and transepithelial electrical resistance (TEER) were used to analyze damage in the Caco2 intestinal barrier model. The Azoxymethane/Dextran Sulfate Sodium (AOM/DSS) animal model was established to evaluate the effect of ETBF on intestinal barrier injury and CRC progression in vivo. ETBF and BFT enhanced the viability, wound healing ratio, invasion, and EMT of SW480 cells. In addition, ETBF and BFT disrupted the tight junctions and villus structure in the intestinal barrier model, resulting in increased permeability and reduced TEER. Similarly, the expression of intestinal barrier-related proteins (MUC2, Occludin and Zo-1) was restricted by ETBF and BFT. Interestingly, the STAT3/ZEB2 axis was activated by ETBF and BFT, and treatment with Brevilin A (a STAT3 inhibitor) or knockdown of ZEB2 limited the promotional effect of ETBF and BFT on the SW480 malignant phenotype. In vivo experiments also confirmed that ETBF colonization accelerated tumor load, carcinogenesis, and intestinal mucosal barrier damage in the colorectum of the AOM/DSS animal model, and that treatment with Brevilin A alleviated these processes. ETBF-secreted BFT accelerated intestinal barrier damage and CRC by activating the STAT3/ZEB2 axis. Our findings provide new insights and perspectives for the application of ETBF in CRC treatment.

Bacteroides fragilis toxin expression enables lamina propria niche acquisition in the developing mouse gut.

Bacterial toxins are well-studied virulence factors; however, recent studies have revealed their importance in bacterial niche adaptation. Enterotoxigenic Bacteroides fragilis (ETBF) expresses B. fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition. We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B. fragilis. LP entry by ETBF required BFT metalloprotease activity, and showed temporal restriction to the pre-weaning period, dependent on goblet-cell-associated passages. In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP. BFT expression increased goblet cell number and goblet-cell-associated passage formation. These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition, suggesting that a selective advantage conferred by a toxin may impact long-term host health.

Enrichment of Bacteroides fragilis and enterotoxigenic Bacteroides fragilis in CpG island methylator phenotype-high colorectal carcinoma

ObjectivesData support that enterotoxigenic Bacteroides fragilis (ETBF) harbouring the Bacteroides fragilis toxin (bft) gene may promote colorectal tumourigenesis through the serrated neoplasia pathway. We hypothesized that ETBF may be enriched in colorectal carcinoma subtypes with high-level CpG island methylator phenotype (CIMP-high), BRAF mutation, and high-level microsatellite instability (MSI-high). MethodsQuantitative PCR assays were designed to quantify DNA amounts of Bacteroides fragilis, ETBF, and each bft gene isotype (bft-1, bft-2, or bft-3) in colorectal carcinomas in the Health Professionals Follow-up Study and Nurses' Health Study. We used multivariable-adjusted logistic regression models with the inverse probability weighting method. ResultsWe documented 4476 colorectal cancer cases, including 1232 cases with available bacterial data. High DNA amounts of Bacteroides fragilis and ETBF were positively associated with BRAF mutation (p ≤ 0.0003), CIMP-high (p ≤ 0.0002), and MSI-high (p < 0.0001 and p = 0.01, respectively). Multivariable-adjusted odds ratios (with 95% confidence interval) for high Bacteroides fragilis were 1.40 (1.06–1.85) for CIMP-high and 2.14 (1.65–2.77) for MSI-high, but 1.02 (0.78–1.35) for BRAF mutation. Multivariable-adjusted odds ratios for high ETBF were 2.00 (1.16–3.45) for CIMP-high and 2.86 (1.64–5.00) for BRAF mutation, but 1.09 (0.67–1.76) for MSI-high. Neither Bacteroides fragilis nor ETBF was associated with colorectal cancer-specific or overall survival. DiscussionThe tissue abundance of Bacteroides fragilis is associated with CIMP-high and MSI-high, whereas ETBF abundance is associated with CIMP-high and BRAF mutation in colorectal carcinoma. Our findings support the aetiological relevance of Bacteroides fragilis and ETBF in the serrated neoplasia pathway.

Promotion of Colitis in B Cell-Deficient C57BL/6 Mice Infected with Enterotoxigenic Bacteroides fragilis.

Enterotoxigenic Bacteroides fragilis (ETBF) causes colitis and is implicated in inflammatory bowel diseases and colorectal cancer. The ETBF-secreted B. fragilis toxin (BFT) causes cleavage of the adherence junction, the E-cadherin, resulting in the large intestine showing IL-17A inflammation in wild-type (WT) mice. However, intestinal pathology by ETBF infection is not fully understood in B-cell-deficient mice. In this study, ETBF-mediated inflammation was characterized in B-cell-deficient mice (muMT). WT or muMT C57BL/6J mice were orally inoculated with ETBF and examined for intestinal inflammation. The indirect indicators for colitis (loss of body weight and cecum weight, as well as mortality) were increased in muMT mice compared to WT mice. Histopathology and inflammatory genes (Nos2, Il-1β, Tnf-α, and Cxcl1) were elevated and persisted in the large intestine of muMT mice compared with WT mice during chronic ETBF infection. However, intestinal IL-17A expression was comparable between WT and muMT mice during infection. Consistently, flow cytometry analysis applied to the mesenteric lymph nodes showed a similar Th17 immune response in both WT and muMT mice. Despite elevated ETBF colonization, the ETBF-infected muMT mice showed no histopathology or inflammation in the small intestine. In conclusion, B cells play a protective role in ETBF-induced colitis, and IL-17A inflammation is not attributed to prompted colitis in B-cell-deficient mice. Our data support the fact that B cells are required to ameliorate ETBF infection-induced colitis in the host.

Antibody response to enterotoxigenic Bacteroides fragilis of Filipino colorectal cancer patients.

Several species of the gut microbiota have been implicated in colorectal cancer (CRC) development. The anaerobic bacterium enterotoxigenic Bacteroides fragilis (ETBF), has been identified to produce fragilysin, a toxin known to cleave E-cadherin, thereby leading to carcinogenesis. To determine the antibody response of CRC patients against ETBF to ascertain whether significant difference exists or whether antibody response is related to tumor grade and tumor stage. Informed consent was obtained from histologically confirmed CRC casesand their age- and sex-matched clinically healthy controls. Plasma samples from the participants were subjected to in-house enzyme-linked immunosorbent assay (ELISA) to determine their antibody levels. Using ETBF total protein as coating antigen, 38/39 (97%) CRC cases and 36/39 (92%) controls showed anti-ETBF IgG above cut-off, while all (100%) CRC cases and 36/39 (92%) controls had anti-ETBF IgA levels above cut-off. With culture broth as coating antigen, all (100%) CRC cases and 37/39 (95%) controls had anti-ETBF IgG levels above cut-off. For anti-ETBF IgA, all (100%) cases and controls had levels above cut-off. Statistical analysis reveals no significant difference (P > 0.05) on the number of CRC cases and controls with IgG and IgA antibody levels above cut-off value. Also, there's no significant difference (P > 0.05) in the mean anti-ETBF antibody levels of cases who were at different tumor grade (well differentiated and moderately and poorly differentiated) and tumor stage (early and advanced). These results suggest that Filipino CRC cases and their clinically healthy matched controls exhibit antibody responses against ETBF.

Gut bacteria promote proliferation in benign S/RG/C2 colorectal tumour cells, and promote proliferation, migration and invasion in malignant HCT116 cells

Colorectal cancer (CRC) is a significant global health burden with a rising incidence worldwide. Distinct bacterial populations are associated with CRC development and progression, and it is thought that the relationship between CRC and associated gut bacteria changes during the progression from normal epithelium to benign adenoma and eventually malignant carcinoma and metastasis. This study compared the interaction of CRC-associated species Enterotoxigenic Bacteroides fragilis, Enterococcus faecalis and Fusobacterium nucleatum and one probiotic species, Escherichia coli Nissle 1917 with a colorectal adenoma (S/RG/C2) and a colorectal adenocarcinoma (HCT116) derived cell line. Gentamicin protection assays showed that all species displayed higher attachment to benign tumour monolayers when compared to malignant monolayers. However, invasion of 3/4 species was higher in the HCT116 cells than in the adenoma cells. All species were found to persist within tumour cell monolayers for a minimum of 48 h under standard aerobic cell culture conditions, with persistence significantly higher in HCT116 cells. Downstream assays were performed to analyse the behaviour of S/RG/C2 and HCT116 cells post-infection and revealed that all species increased the tumour cell yield of both cell lines. The migratory and invasive potential of HCT116 cells was increased after infection with F. nucleatum; however, no species significantly altered these characteristics in S/RG/C2 cells. These results add to the growing evidence for the involvement of microorganisms in CRC progression and suggest that these interactions may be dependent on tumour cell-specific characteristics.

Bacteroides fragilis Enterotoxin Induces Autophagy through an AMPK and FoxO3-Pathway, Leading to the Inhibition of Apoptosis in Intestinal Epithelial Cells.

Macroautophagy/autophagy is essential for preserving cellular homeostasis by recycling nutrients and removing spoiled or aged proteins and organelles. It also has an essential role in defense mechanisms against microbial infections. However, the role of autophagy in enterotoxigenic Bacteroides fragilis infection remains largely unknown. In this study, we explored the role of B. fragilis enterotoxin (BFT) in the autophagic process of intestinal epithelial cells (IECs). The LC3-I of human HCT-116 IECs was converted to LC3-II by BFT stimulation. In addition, BFT-exposed cells showed the decreased expression of p62 in a time-dependent manner and increased levels of ATG5 and ATG12 gradually. Evidence of an enhanced autophagic process was supported by autophagosomes co-localized with LC3-lysosome-associated protein 2 in BFT-stimulated cells. The AMP-activated protein kinase (AMPK) and Forkhead box O3 (FoxO3a) axis were required for BFT-induced autophagy activation. In contrast with the activation of autophagy at 3-6 h after BFT exposure, IECs induced apoptosis-related signals at 12-48 h. HCT-116 IECs suppressing the formation of autophagosomes significantly activated apoptosis signals instead of autophagy early after BFT exposure. These data suggest that BFT can activate autophagy through the AMPK-FoxO3a pathway and the autophagy may suppress apoptosis during early exposure of IECs to BFT.

Evaluation of enterotoxigenic Bacteroides fragilis correlation with the expression of cellular signaling pathway genes in Iranian patients with colorectal cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers all over the world, and dysbiosis in the gut microbiota may play a role in colorectal carcinogenesis. Bacteroides fragilis can lead to tumorigenesis by changing signaling pathways, including the WNT/β-catenin pathway. Therefore, in the present study, we investigated the correlation between the enterotoxigenic B. fragilis amount and the expression of signaling pathway genes involved in CRC.Materials and methodsB. fragilis was determined in 30 tumors and adjacent healthy tissues by the qPCR method. Next, the relationship between enterotoxigenic B. fragilis and the expression of signaling pathway genes, including CCND1, TP53, BCL2, BAX, WNT, TCF, AXIN, APC, and CTNNB1 was investigated. Additionally, possible correlations between clinicopathological features of the tumor samples and the abundance of B. fragilis were analyzed.ResultsThe results showed that B. fragilis was detected in 100% of tumor samples and 86% of healthy tissues. Additionally, enterotoxigenic B. fragilis colonized 47% of all samples, and bft-1 toxin was the most frequently found isotype among the samples. The analysis showed that the high level of B. fragilis has a significant relationship with the high expression of AXIN, CTNNB1, and BCL2 genes. On the other hand, our results did not show any possible correlation between this bacterium and the clinicopathological features of the tumor sample.ConclusionB. fragilis had a higher abundance in the tumor samples than in healthy tissues, and this bacterium may lead to CRC by making changes in cellular signaling pathways and genes. Therefore, to better understand the physiological effects of B. fragilis on the inflammatory response and CRC, future research should focus on dissecting the molecular mechanisms by which this bacterium regulates cellular signaling pathways.

Hepatobiliary manganese homeostasis is dynamic in the setting of inflammation or infection in mice.

Manganese is a diet-derived micronutrient that is essential for critical cellular processes like redox homeostasis, protein glycosylation, and lipid and carbohydrate metabolism. Control of Mn availability, especially at the local site of infection, is a key component of the innate immune response. Less has been elucidated about Mn homeostasis at the systemic level. In this work, we demonstrate that systemic Mn homeostasis is dynamic in response to inflammation and infection in mice. This phenomenon is evidenced in male and female mice, mice of two genetic backgrounds (C57BL/6 and BALB/c), in multiple models of acute (dextran sodium sulfate-induced) and chronic (enterotoxigenic Bacteroides fragilis) colitis, and systemic infection with Candida albicans. When mice were fed a standard corn-based chow with excess Mn (100 ppm), liver Mn decreased and biliary Mn increased threefold in response to infection or colitis. Liver iron, copper, and zinc were unchanged. When dietary Mn was restricted to minimally adequate amounts (10 ppm), baseline hepatic Mn levels decreased by approximately 60% in the liver, and upon induction of colitis, liver Mn did not decrease further, however, biliary Mn still increased 20-fold. In response to acute colitis, hepatic Slc39a8 mRNA (gene encoding the Mn importer, Zip8) and Slc30a10 mRNA (gene encoding the Mn exporter, Znt10) are decreased. Zip8 protein is decreased. Inflammation/infection-associated dynamic Mn homeostasis may represent a novel host immune/inflammatory response that reorganizes systemic Mn availability through differential expression of key Mn transporters with down-regulation of Zip8.

The Relationship of Enterotoxigenic Bacteroides fragilis and Fusobacterium nucleatum Intestinal Colonization with Colorectal Cancer: A Case-Control Study Performed with Colon Biopsies

In recent years, it has been shown that some bacteria may be associated with colorectal cancer (CRC). In this study, it was aimed to investigate the role of Fusobacterium nucleatum and enterotoxigenic Bacteroides fragilis (ETBF) in the etiology of CRC by comparing the amounts of these bacteria in colon biopsy tissues of patients with CRC and healthy individuals. The amounts of F.nucleatum and ETBF were determined by quantitative polymerase chain reaction (qPCR) in colon biopsy samples taken from 35 CRC and 35 healthy individuals, and the results were compared in the patient and control groups. The detection rate and amounts of F.nucleatum were found to be statistically significantly higher in tissues of female patients with CRC compared to male patients (p= 0.003, p= 0.013, respectively). There was no statistically significant difference between the tissues of female and male patients with CRC in terms of detection rate and amount of ETBF (p= 0.521, p= 0.515, respectively). It was found that in the 50-74 age group, the amount of ETBF was statistically significantly higher in women and men with CRC compared to the controls (p= 0.005, p= 0.047, respectively), while the amount of F.nucleatum was statistically significantly higher in female patients compared to controls. However, no difference was found between male patients and controls (p= 0.009, p= 0.083). It was determined that the detection rate and amount of F.nucleatum in the tissues of patients with CRC, regardless of age and gender, were not statistically different from the controls (p= 0.473, p= 0.995, respectively), however, the detection rate of ETBF and the amount of ETBF were found to be statistically significantly higher (p= 0.002, p= 0.004, respectively). It has been determined that ETBF can play a role in the etiology of CRC in both men and women, and F.nucleatum only in women, in the age range of 50-74 years, when routine screenings for CRC are performed.

Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis

ObjectiveThe aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. MethodsETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. ResultsSix different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). ConclusionsOur data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

Abstract 5905: Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression

Abstract Background: With more than 2.3 million cases worldwide, breast cancer persists as a major health burden and warrants a greater understanding of the molecular triggers underlying this disease. Focusing on microbial dysbiosis, our lab discovered the pivotal role of the enterotoxigenic Bacteroides fragilis (ETBF) in promoting breast tumorigenesis through activation of key oncogenic pathways. A gap in knowledge exists regarding how microbes/microbial toxins modulate the oncogenic pathways in cancer cells. Polyamines are imperative for various physiological processes, including cell growth, survival, differentiation and apoptosis and are strongly associated with breast cancer. We questioned the involvement of polyamine pathway in mediating the oncogenic effects of microbial dysbiosis on breast cancer. Methods: Human Her2 expressing spontaneous breast cancer model, MMTV.f.HuHer2 mice, were infected with ETBF and tumor incidence and progression were monitored for 18 months. We used qRT-PCR, western blotting, immunocytochemistry and immunohistochemistry to examine expression of key nodes of polyamine pathway. Various growth, migration and invasion assays were performed to assess carcinogenic properties in breast cancer cells. We employed a colorimetric-based assay to analyze enzymatic activities of key polyamine metabolic members. ROS generation was examined via DCFA staining. Results: Our in vivo data showed that ETBF colonization in mice led to a considerable increase in the level of spermine oxidase (SMOX) in breast tumors, hinting at a correlation between ETBF and polyamine metabolism. Moreover, a marked rise in expression of γH2AX foci was observed in these tumors and in breast cancer cells, suggesting a critical role of ETBF in SMOX-related DNA damage. In agreement, we found SMOX upregulation in breast cancer cells. Our findings displayed a substantial increase in SMOX enzymatic activity in the presence of ETBF toxin, BFT, in breast tumor cells. BFT caused a marked rise in ROS generation of breast tumor cells, suggesting a plausible association of ETBF in SMOX-mediated oxidative stress. Interestingly, we noted a significant induction of ornithine decarboxylase (ODC) expression and activity in response to ETBF or BFT in breast cancer cells. Treatment of BFT-exposed breast cancer cells with SMOX inhibitors led to a sharp inhibition of neoplastic transformations, like cellular migration and γH2AX induction. Conclusion: Our collated evidences suggest the existence of an important cross-talk between key polyamine metabolic enzymes and ETBF that aids in exacerbating breast tumorigenesis. Of importance, therapeutic inhibition of SMOX dramatically impeded BFT-related breast tumorigenesis. Collectively, our results demonstrated the attractive potential of intervening the polyamine catabolic pathway for the management of breast cancer. Citation Format: Deeptashree Nandi, Sheetal Parida, Sowjanya Thatikonda, Jackson Foley, Tracy Stewart, Cynthia L. Sears, Robert A. Casero, Dipali Sharma. Enterotoxigenic Bacteroides fragilis, a colon microbe, dysregulates polyamine catabolism to promote breast cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5905.

Abstract 3039: Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases

Abstract Background: Colorectal cancer (CRC) incidence has increased in those 50 years or younger compared to older individuals in recent decades. While the intra-tumoral microbiome is a potent facilitator of tumorigenesis, the role of the microbiome in early onset (eo)CRC is unclear. Identification of the intra-tumoral microbiome may enhance prediction of adverse outcomes to improve survival of CRC overall age groups and eoCRC. Methods: We performed targeted sequencing of tumor and matched normal DNA samples from 3695 colorectal cancer cases within the Genetics and Colorectal Cancer Consortium (GECCO) and the Colon Cancer Family Registry (CCFR). Tumor DNA was extracted from formalin fixed paraffin embedded (FFPE) or fresh frozen tissue. Normal tissue DNA was extracted from blood or normal surrounding colorectal tissue. We designed probes for the bacterial pathogens enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase positive E. coli (pks+ EC), and Fusobacterium nucleatum (Fn). We used logistic regression to assess tumor-associated bacteria and clinical parameters, tumor molecular characteristics, and tumor stage adjusted for age, sex, and study and a Cox promotional hazard to assess association with CRC-specific survival, overall and in eoCRC, adjusted for age at diagnosis, sex, mutational burden, and microsatellite instability (MSI) and stratified baseline hazards by study. Survival data were available for 2357 and 308 patients, overall and in eoCRC, respectively. Results: The prevalence of different bacteria in tumors differed slightly overall vs. in eoCRC tumors: ETBF (5.2%, 6.3%), pks+ EC (12.9%, 14.8%), and Fn (10.4%, 8.2%). Overall, men had a lower probability of tumors with Fn (odds ratio (OR)=0.8, 95% confidence interval (CI)=0.64-0.99, p=0.04) but higher odds of tumors with pks+ EC (OR=1.38, CI=1.14-1.68, p=0.001), compared to women. The odds of prevalent Fn were higher for proximal vs distal tumors (OR=1.76, CI=1.38-2.26, p&amp;lt;0.001), but there was no association between tumor site and pks+ EC or ETBF. MSI-high tumors were more likely to carry Fn (OR=2.79, CI=2.08-3.75, p&amp;lt;0.001) and less likely to carry pks+ EC (OR=0.63, CI=0.44-0.88, p=0.008) when compared to patients with MSS tumors. In patients with non-hypermutated tumors, we observed that pks+ EC positive tumors were associated with better CRC-specific survival (HR=0.73, CI=0.57-0.93, p=0.01), whereas ETBF positive tumors (HR=1.99, CI=1,18-3.36,p=0.009) and Fn positive tumors (HR=1.33, CI=1.04-1.72, p=0.03) were associated with poorer survival. In eo-CRC patients, Fn positive status was associated with poorer survival (HR=2.4, CI= 1.056 - 5.492, p=0.026). Conclusions: We showed that the intra-tumoral microbiome differs in distribution across patient populations and patient survival over all age groups and, specifically, in eoCRC. Citation Format: Meredith A. Hullar, Keith R. Curtis, Tabitha Harrison, Yi Lin, Robert Steinfelder, Sonja I. Berndt, Daniel D. Buchanan, Andrew T. Chan, David A. Drew, Jane Figueiredo, Amy J. French, Tabitha A. Harrison, Mark A. Jenkins, Johannes Melaku, Victor Moreno, Tomotaka Ugai, Shuji Ogino, Conghui Qu, Chenxu Qu, Steven Thibodeau, Syed Zaidi, Amanda I. Phipps, Ulrike Peters, on behalf of GECCO and CCFR investigators. Evaluation of intra-tumoral pks+ E. coli, enterotoxigenic B. fragilis and Fusobacterium nucleatum, overall and in early onset disease, in colorectal cancer cases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3039.