1254a
ACTIVATING TRANSCRIPTION FACTOR 6 ACCELERATES MICROBIOTA-DEPENDENT RISK FOR COLITIS-ASSOCIATED CANCER IN INFLAMMATORY BOWEL DISEASES
Date
May 9, 2023
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Society: AGA
BACKGROUND AND AIMS: Chemoresistance is a major cause of colorectal cancer (CRC) recurrence and death. The critical role of gut microbiome in the efficacy of CRC chemotherapy remains unclear. We aim to characterize the gut microbiota signatures in responders and non-responders to chemotherapy and identify chemoresistance-associated pathogens in CRC.
METHODS: Stool samples were collected from CRC patients before chemotherapy (n=64) and analyzed by shotgun metagenomic sequencing. The effect of enterotoxigenic Bacteroides fragilis (ETBF) on chemoresistance was assessed in CRC cells and CRC mouse models of xenografts, ApcMin/+ or azoxymethane (AOM)/dextran sodium sulfate (DSS) induced CRC tumorigenesis. Phage VA7 was used for targeting ETBF in vitro and in vivo. Bacterial adhesin-host receptor interaction was investigated by far western, biotin pull-down, and GST pull-down assays followed by mass spectrometry. RNA-seq was performed to evaluate involved pathways.
RESULTS: Bacteroides fragilis was identified as the top enriched bacterium in stool samples of CRC patients who were resistant to chemotherapy, and its abundance was associated with poor prognosis (P=0.019). Consistent with this observation, we demonstrated that ETBF co-culture with CRC cells (HT29 and HCT116) inhibited 5-FU and oxaliplatin (OXA)-induced cell apoptosis (both P<0.001). ETBF promoted chemoresistance in xenografts of nude mice (P<0.001). In particular, oral gavage of ETBF to ApcMin/+ mice significantly attenuated the antitumor efficacy of 5-FU and OXA. Such effect was further confirmed in the second CRC tumorigenesis mouse model induced by AOM/DSS. Mechanistically, we revealed that ETBF activated Notch signaling pathway genes (Notch1, NICD, Hes1, MAML1, and c-Myc) identified by RNA-seq and confirmed by Western blot. ETBF-mediated chemoresistance critically depends on its direct interaction with CRC cells, as neither ETBF supernatant nor non-contact transwell co-culture significantly affected the efficacy of 5-FU and OXA. Concordantly, fluorescence in situ hybridization (FISH) and qPCR demonstrated the significant enrichment of ETBF in colonic tumors compared to adjacent normal tissues (P = 0.002) in mice. Scanning electron microscopy (SEM) visualized the attachment of ETBF to colon epithelial cells. ETBF surface protein SusD/RagB was identified to mediate its attachment to CRC cells. Finally, we demonstrated that ETBF-targeting phage VA7 selectively inhibited ETBF in vitro and in mice (P<0.05), and that VA7 co-administration abolished ETBF-induced chemoresistance (P<0.001).
CONCLUSION: We identified for the first time that ETBF provokes chemoresistance in CRC cells through its direct interaction with colon epithelial cells and activating Notch1 signaling. Targeting ETBF by phage VA7 is a promising therapeutic strategy to overcome ETBF-mediated chemoresistance in CRC.
METHODS: Stool samples were collected from CRC patients before chemotherapy (n=64) and analyzed by shotgun metagenomic sequencing. The effect of enterotoxigenic Bacteroides fragilis (ETBF) on chemoresistance was assessed in CRC cells and CRC mouse models of xenografts, ApcMin/+ or azoxymethane (AOM)/dextran sodium sulfate (DSS) induced CRC tumorigenesis. Phage VA7 was used for targeting ETBF in vitro and in vivo. Bacterial adhesin-host receptor interaction was investigated by far western, biotin pull-down, and GST pull-down assays followed by mass spectrometry. RNA-seq was performed to evaluate involved pathways.
RESULTS: Bacteroides fragilis was identified as the top enriched bacterium in stool samples of CRC patients who were resistant to chemotherapy, and its abundance was associated with poor prognosis (P=0.019). Consistent with this observation, we demonstrated that ETBF co-culture with CRC cells (HT29 and HCT116) inhibited 5-FU and oxaliplatin (OXA)-induced cell apoptosis (both P<0.001). ETBF promoted chemoresistance in xenografts of nude mice (P<0.001). In particular, oral gavage of ETBF to ApcMin/+ mice significantly attenuated the antitumor efficacy of 5-FU and OXA. Such effect was further confirmed in the second CRC tumorigenesis mouse model induced by AOM/DSS. Mechanistically, we revealed that ETBF activated Notch signaling pathway genes (Notch1, NICD, Hes1, MAML1, and c-Myc) identified by RNA-seq and confirmed by Western blot. ETBF-mediated chemoresistance critically depends on its direct interaction with CRC cells, as neither ETBF supernatant nor non-contact transwell co-culture significantly affected the efficacy of 5-FU and OXA. Concordantly, fluorescence in situ hybridization (FISH) and qPCR demonstrated the significant enrichment of ETBF in colonic tumors compared to adjacent normal tissues (P = 0.002) in mice. Scanning electron microscopy (SEM) visualized the attachment of ETBF to colon epithelial cells. ETBF surface protein SusD/RagB was identified to mediate its attachment to CRC cells. Finally, we demonstrated that ETBF-targeting phage VA7 selectively inhibited ETBF in vitro and in mice (P<0.05), and that VA7 co-administration abolished ETBF-induced chemoresistance (P<0.001).
CONCLUSION: We identified for the first time that ETBF provokes chemoresistance in CRC cells through its direct interaction with colon epithelial cells and activating Notch1 signaling. Targeting ETBF by phage VA7 is a promising therapeutic strategy to overcome ETBF-mediated chemoresistance in CRC.
Background
Diet interacts intimately with the gut microbiome and is also a major risk factor for colorectal cancer (CRC). However, epidemiologic evidence on how diet may influence CRC through alteration of the gut microbiome remains limited.
Methods
We leveraged two cohorts of 307 healthy men and 212 healthy women with serial stool metagenomes and detailed dietary data. We empirically characterized a dietary pattern associated with the CRC gut microbiome signature (labeled CRC Microbial Dietary Score [CRC-MDS]), which sums the abundances of 30 species enriched and 13 depleted in CRC based on their loadings in a recent meta-analysis of metagenomic studies (Thomas et al. Nat Med. 2019). We then applied the CRC-MDS to three prospective cohorts – the Health Professionals Follow-up Study (1986-2014), the Nurses’ Health Study (1984-2014), and the Nurses’ Health Study II (1991-2015), in which we assessed diet by semi-quantitative food frequency questionnaires every four years, confirmed self-reported CRC cases through medical record review, and collected tumor tissues from a subset of CRC patients. We evaluated the CRC-MDS association with CRC risk using Cox proportional hazards model, and further assessed CRC risk according to subsites and tumor tissue presence of Fusobacterium nucleatum (F. nucleatum).
Results
The CRC-MDS was characterized by high consumption of foods with greater industrial processing (e.g., processed meat, butter, and low/high-energy drinks) and low consumption of foods rich in fiber (e.g., fruits, vegetables, nuts, and whole grains) (Figure). The score was positively correlated with Western diet (Spearman ρ =0.44) and empirical dietary index for hyperinsulinemia score (ρ =0.54), and negatively correlated with the prudent dietary pattern score (ρ =-0.53). In the three cohorts of 259,190 participants, we documented 3,819 incident cases of CRC over 3,223,696 person-years of follow-up. Compared with participants in the lowest quintile of the CRC-MDS, those in the highest quintile had an increased risk of CRC (hazard ratio [HR], 1.25; 95% confidence interval [CI], 1.12-1.39; Ptrend <0.001) after adjusting for putative CRC risk factors. The association was attenuated but remained statistically significant after adjusting for other dietary patterns (see details in Table). The association was similar across CRC subsites but differed according to the presence of F. nucleatum in tumor tissue. The CRC-MDS was strongly associated with a higher risk of F. nucleatum-positive CRC (HRQ5 vs Q1, 2.52; 95% CI, 1.68-3.76; Ptrend <0.001) but not F. nucleatum-negative CRC (HRQ5 vs Q1, 1.09; 95% CI, 0.95-1.25; Ptrend =0.19) (Pheterogeneity = 0.03).
Conclusions
The CRC-MDS was positively associated with CRC risk, especially F. nucleatum-positive CRC. Our findings support the gut microbiome as a mediator of diet in CRC development.
Diet interacts intimately with the gut microbiome and is also a major risk factor for colorectal cancer (CRC). However, epidemiologic evidence on how diet may influence CRC through alteration of the gut microbiome remains limited.
Methods
We leveraged two cohorts of 307 healthy men and 212 healthy women with serial stool metagenomes and detailed dietary data. We empirically characterized a dietary pattern associated with the CRC gut microbiome signature (labeled CRC Microbial Dietary Score [CRC-MDS]), which sums the abundances of 30 species enriched and 13 depleted in CRC based on their loadings in a recent meta-analysis of metagenomic studies (Thomas et al. Nat Med. 2019). We then applied the CRC-MDS to three prospective cohorts – the Health Professionals Follow-up Study (1986-2014), the Nurses’ Health Study (1984-2014), and the Nurses’ Health Study II (1991-2015), in which we assessed diet by semi-quantitative food frequency questionnaires every four years, confirmed self-reported CRC cases through medical record review, and collected tumor tissues from a subset of CRC patients. We evaluated the CRC-MDS association with CRC risk using Cox proportional hazards model, and further assessed CRC risk according to subsites and tumor tissue presence of Fusobacterium nucleatum (F. nucleatum).
Results
The CRC-MDS was characterized by high consumption of foods with greater industrial processing (e.g., processed meat, butter, and low/high-energy drinks) and low consumption of foods rich in fiber (e.g., fruits, vegetables, nuts, and whole grains) (Figure). The score was positively correlated with Western diet (Spearman ρ =0.44) and empirical dietary index for hyperinsulinemia score (ρ =0.54), and negatively correlated with the prudent dietary pattern score (ρ =-0.53). In the three cohorts of 259,190 participants, we documented 3,819 incident cases of CRC over 3,223,696 person-years of follow-up. Compared with participants in the lowest quintile of the CRC-MDS, those in the highest quintile had an increased risk of CRC (hazard ratio [HR], 1.25; 95% confidence interval [CI], 1.12-1.39; Ptrend <0.001) after adjusting for putative CRC risk factors. The association was attenuated but remained statistically significant after adjusting for other dietary patterns (see details in Table). The association was similar across CRC subsites but differed according to the presence of F. nucleatum in tumor tissue. The CRC-MDS was strongly associated with a higher risk of F. nucleatum-positive CRC (HRQ5 vs Q1, 2.52; 95% CI, 1.68-3.76; Ptrend <0.001) but not F. nucleatum-negative CRC (HRQ5 vs Q1, 1.09; 95% CI, 0.95-1.25; Ptrend =0.19) (Pheterogeneity = 0.03).
Conclusions
The CRC-MDS was positively associated with CRC risk, especially F. nucleatum-positive CRC. Our findings support the gut microbiome as a mediator of diet in CRC development.
Figure. Weights for food groups comprising the CRC-MDS in men (A) and women (B)
Table. Hazard ratios (95% CIs) for colorectal cancer according to the CRC-MDS
Background: Invasive bacterial biofilms are associated with irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colorectal cancer (CRC) particularly in the proximal colon, but limited data is available on their prevalence on histologically normal colonic tissues or polyps. The present study examined biofilms in a healthy screening colonoscopy cohort to determine the variability of mucosal biofilms along the colonic axis in normal tissues and in early colonic lesions.
Methods: Biofilms were examined in a screening colonoscopy cohort of 2,091 individuals from three sites in Maryland and/or Pennsylvania, USA. Six methacarn-fixed, histologically normal colonic biopsies per patient (3 left, 3 right colon) were evaluated by fluorescence in situ hybridization (FISH) with the EUB338 probe on a scale from 0-3 (3 being a biofilm, with >20 adherent bacteria within 1 mm of the epithelium along a 200 mm stretch). Patients with IBD and patients on blood thinners were ineligible for the study. Inclusion criteria for the present analysis included patients without a history of CRC or new-onset CRC, and optimally acquired biopsy samples as previously reported (large capacity biopsy forceps and >7 hours time from bowel prep completion to the start of colonoscopy) (N = 1,226). Forty-one paired, formalin-fixed polyp tissues from 21 individuals (7 biofilm positive, 14 biofilm negative on normal mucosa) were also screened for biofilms.
Results: A total of 11.3% (138/1,226) of patients were scored as biofilm positive on their histologically normal tissue. A high degree of intra-individual variability was observed, with only 29/138 biofilm-positive patients having biofilms on both right and left colonic biopsies. Biofilms on histologically normal tissues were not associated with polyp status using linear regression modeling (see Rifkin et al, DDW 2023 abstract). While a similar prevalence and distribution of biofilms was observed on polyps vs. histologically normal tissues (10.5% vs. 11.3%, respectively), the polyp bacterial scores overall were nearly double that of the paired, normal tissues (p = 0.0005, Mann-Whitney, median score of 2 on polyps vs. 1.15 on normal tissues). This difference was weighted strongly by biofilm-negative individuals (Figure 1A, p < 0.0001). Polyp bacterial scores significantly correlated with polyp size (p = 0.014, Spearman’s rank correlation). Furthermore, several large polyps displayed dramatic bacterial crypt invasion that, while still separated from the epithelium by mucus (Figure 1B), were reminiscent of the prolific invasion seen in colorectal cancer specimens.
Conclusions: Colonic biofilms are dynamic ecosystems that may be transient and focal in healthy mucosa but appear to co-evolve with early colonic lesions along the adenoma-carcinoma sequence and may facilitate progression to colorectal cancer.
Methods: Biofilms were examined in a screening colonoscopy cohort of 2,091 individuals from three sites in Maryland and/or Pennsylvania, USA. Six methacarn-fixed, histologically normal colonic biopsies per patient (3 left, 3 right colon) were evaluated by fluorescence in situ hybridization (FISH) with the EUB338 probe on a scale from 0-3 (3 being a biofilm, with >20 adherent bacteria within 1 mm of the epithelium along a 200 mm stretch). Patients with IBD and patients on blood thinners were ineligible for the study. Inclusion criteria for the present analysis included patients without a history of CRC or new-onset CRC, and optimally acquired biopsy samples as previously reported (large capacity biopsy forceps and >7 hours time from bowel prep completion to the start of colonoscopy) (N = 1,226). Forty-one paired, formalin-fixed polyp tissues from 21 individuals (7 biofilm positive, 14 biofilm negative on normal mucosa) were also screened for biofilms.
Results: A total of 11.3% (138/1,226) of patients were scored as biofilm positive on their histologically normal tissue. A high degree of intra-individual variability was observed, with only 29/138 biofilm-positive patients having biofilms on both right and left colonic biopsies. Biofilms on histologically normal tissues were not associated with polyp status using linear regression modeling (see Rifkin et al, DDW 2023 abstract). While a similar prevalence and distribution of biofilms was observed on polyps vs. histologically normal tissues (10.5% vs. 11.3%, respectively), the polyp bacterial scores overall were nearly double that of the paired, normal tissues (p = 0.0005, Mann-Whitney, median score of 2 on polyps vs. 1.15 on normal tissues). This difference was weighted strongly by biofilm-negative individuals (Figure 1A, p < 0.0001). Polyp bacterial scores significantly correlated with polyp size (p = 0.014, Spearman’s rank correlation). Furthermore, several large polyps displayed dramatic bacterial crypt invasion that, while still separated from the epithelium by mucus (Figure 1B), were reminiscent of the prolific invasion seen in colorectal cancer specimens.
Conclusions: Colonic biofilms are dynamic ecosystems that may be transient and focal in healthy mucosa but appear to co-evolve with early colonic lesions along the adenoma-carcinoma sequence and may facilitate progression to colorectal cancer.
Figure 1. Polyps display higher bacterial scores and more pronounced bacterial invasion than paired normal tissues. Forty-one polyps from 21 individuals (range: 1-4 polyps per patient) and their paired normal tissues were screened for bacterial invasion on a scale from 0-3 (3 = biofilm positive). (A) Polyps from biofilm-negative patients, as determined by biofilm status on their normal tissue, displayed higher bacterial scores than their paired normal tissues. Polyps from biofilm-positive patients were equivalent to their biofilm-positive normal tissues but were still significantly higher than polyps from biofilm-negative patients. Mann-Whitney p-values are shown. (B) A formalin-fixed, transverse tubular adenoma from a biofilm-positive patient presented with prolific crypt infiltration throughout the tissue (white arrows). Blue = DAPI, yellow = EUB338, green = autofluorescence.
Introduction: Chronic inflammation in the colon of patients with inflammatory bowel diseases (IBD) represents a risk factor for developing colitis-associated cancer (CAC). Activating transcription factor 6 (ATF6) signaling provides a novel link between the endoplasmic reticulum unfolded protein response (UPRER) of the epithelium and the development of spontaneous colorectal cancer (CRC). In this study, we characterize the impact of IBD-related risk factors on ATF6-mediated tumorigenesis in gnotobiotic mouse models and patients.
Methods: We generated a novel transgenic mouse model for spontaneous CAC by crossing mice expressing the active form of ATF6 (nATF6) in intestinal epithelial cells (IEC) with Interleukin-10 deficient mice (nATF6IEC,Il10-/-). Specific pathogen-free (SPF) and germ-free (GF) mice were characterized for tumor formation (incidence, frequency and histology), inflammation (gene expression analysis, immunostaining and immune cell infiltration) and mucus properties (mucus staining and FISH). Spatial microbial signatures of tumor and tumor-associated tissue regions were analyzed by 16S rRNA amplicon sequencing under SPF conditions as well as fecal microbial transfer (FMT) of IBD patient stool, and IBD-relevant simplified human microbiota (SIHUMI). ATF6 expression patterns were quantified in IBD patient tissue.
Results: Constitutive expression of nATF6 drives CAC in Il10-/- mice. Strikingly, tumor-free monoallelic nATF6IEC mice (tg/wt;+/+) develop colonic tumors with an incidence of 70% in the absence of Il10 (tg/wt;-/-). tg/tg;-/- mice never exceed a lifespan of 10 weeks. FACS and CHIP cytometry analyses show neutrophil and monocyte infiltrates together with a decrease in NK cells in the tumor niche. tg/wt;-/- mice display a loss of mucus and increased bacterial penetration of the mucus layer. Spatial analyses show that the mucosa-associated microbiota predicts the tumorigenic phenotype, with alterations restricted to tumor sites. GF mice remain tumor-free. IBD patient FMT into GF mice restores the tumorigenic phenotype in susceptible mice (75% tumor incidence). Interestingly, even an IBD-relevant minimal consortium (SIHUMI) induces colonic tumors with an incidence of 64%. Staining in IBD patients reveals higher ATF6 expression in active disease compared to remission.
Conclusion: Our novel CAC mouse model shows that IBD susceptibility increases the risk to develop ATF6-driven tumors. Tumorigenesis is microbiota-dependent, with mucosa-associated microbiota predicting tumorigenic phenotype. Both IBD patient FMT and SIHUMI successfully transfer the disease phenotype and validate tumor risk. Moreover, IBD patients with active disease show increased ATF6 expression, confirming its clinical relevance. Our findings clearly identify ATF6 as a driver of CAC, suggesting its suitability as a biomarker for cancer development in IBD patients.
Methods: We generated a novel transgenic mouse model for spontaneous CAC by crossing mice expressing the active form of ATF6 (nATF6) in intestinal epithelial cells (IEC) with Interleukin-10 deficient mice (nATF6IEC,Il10-/-). Specific pathogen-free (SPF) and germ-free (GF) mice were characterized for tumor formation (incidence, frequency and histology), inflammation (gene expression analysis, immunostaining and immune cell infiltration) and mucus properties (mucus staining and FISH). Spatial microbial signatures of tumor and tumor-associated tissue regions were analyzed by 16S rRNA amplicon sequencing under SPF conditions as well as fecal microbial transfer (FMT) of IBD patient stool, and IBD-relevant simplified human microbiota (SIHUMI). ATF6 expression patterns were quantified in IBD patient tissue.
Results: Constitutive expression of nATF6 drives CAC in Il10-/- mice. Strikingly, tumor-free monoallelic nATF6IEC mice (tg/wt;+/+) develop colonic tumors with an incidence of 70% in the absence of Il10 (tg/wt;-/-). tg/tg;-/- mice never exceed a lifespan of 10 weeks. FACS and CHIP cytometry analyses show neutrophil and monocyte infiltrates together with a decrease in NK cells in the tumor niche. tg/wt;-/- mice display a loss of mucus and increased bacterial penetration of the mucus layer. Spatial analyses show that the mucosa-associated microbiota predicts the tumorigenic phenotype, with alterations restricted to tumor sites. GF mice remain tumor-free. IBD patient FMT into GF mice restores the tumorigenic phenotype in susceptible mice (75% tumor incidence). Interestingly, even an IBD-relevant minimal consortium (SIHUMI) induces colonic tumors with an incidence of 64%. Staining in IBD patients reveals higher ATF6 expression in active disease compared to remission.
Conclusion: Our novel CAC mouse model shows that IBD susceptibility increases the risk to develop ATF6-driven tumors. Tumorigenesis is microbiota-dependent, with mucosa-associated microbiota predicting tumorigenic phenotype. Both IBD patient FMT and SIHUMI successfully transfer the disease phenotype and validate tumor risk. Moreover, IBD patients with active disease show increased ATF6 expression, confirming its clinical relevance. Our findings clearly identify ATF6 as a driver of CAC, suggesting its suitability as a biomarker for cancer development in IBD patients.
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