Introduction

Although Fusobacterium nucleatum (Fn) is one of the best-characterized pathogenic bacteria in colorectal cancer (CRC), its mechanistic role in driving CRC needs to be further elucidated. We observed the upregulation of cholesterol synthesis enzymes by Fn in vitro. And reduced serum cholesterol by butyrate supplementation has been observed in animal studies [1].

Aims & Methods

This study aims to investigate the roles of Fn and butyrate/butyrate-producing bacteria in regulating host cell cholesterol homeostasis. Three colon cancer cell lines (HT-29, HCT116 and Caco-2) were co-cultured with Fn for four hours, and then treated with culture supernatants of two butyrate-producing bacteria (5% v/v), butyrate (1 mM) or broth control for another 48 to 96 hours after the removal of Fn. The biological functions were determined by cell viability assay, colony formation, wound-healing migration and matrigel invasion assays. Cell cycle was analysed by flow cytometry. Apoptosis was analysed by flow cytometry and TUNEL staining. Protein levels were assessed by western blot and immunostaining. Intracellular cholesterol level was assessed by cell-based cholesterol assay kit. Molecular mechanism was investigated by RNA sequencing and Gene Set Enrichment Analysis (GSEA).

Results

Fn significantly increased cell growth, colony formation, migration and invasion abilities and reduced apoptosis of all three colon cancer cells, while follow-up butyrate-related treatments suppressed the Fn effects on the cells. RNA sequencing and GSEA analyses identified differentially expressed genes significantly enriched in cholesterol synthesis/metabolism pathways (normalized enrichment score >1.5, P<0.05 and FDR <0.15), including cholesterol biosynthesis pathway, cholesterol metabolism, cholesterol synthesis disorders, steroid biosynthesis, statin inhibition of cholesterol production, cholesterol metabolism with Bloch and Kandutsch-Russell pathways, and SREBF and miR33 in cholesterol and lipid homeostasis. Leading edge analysis identified key genes involved in at least four of these pathways, including FDFT1 (Farnesyl-Diphosphate Farnesyltransferase 1), SQLE (squalene epoxidase), HMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1), IDI1 (isopentenyl-diphosphate delta isomerase 1), MSMO1 (methylsterol monooxygenase 1), SC5D (sterol-C5-desaturase), and DHCR7 (7-dehydrocholesterol reductase). All these genes encode key enzymes driving cholesterol biosynthesis and were significantly upregulated by Fn in all cells, as well as SREBP2 (sterol regulatory element binding protein-2) encoding a key transcription factor controlling cholesterol biosynthesis. Interestingly, these pathways and genes were downregulated by butyrate-related treatments. Western blotting confirmed the upregulated expression of SREBP2 and two rate-limiting enzymes in cholesterol biosynthesis (SQLE and DHCR7), which were downregulated by butyrate-related treatments. Intracellular cholesterol was found to be significantly increased by Fn and further reduced by butyrate-related treatments. Total serum cholesterol level was significantly increased in Fn-gavaged mice as compared to sham-treated mice, same as reported previously [2], accompanied by the upregulation of Srebp2, Sqle and Dhcr7 in colonic epithelial cells of Fn-treated mice as compared with sham treatment.

Conclusion

Fn exerts its oncogenic effects partly via upregulating SREBP2 and further activation of cholesterol biosynthesis, which may be suppressed by butyrate supplementation.

References

1. Amiri P, Hosseini SA, Ghaffari S, Tutunchi H, Ghaffari S, Mosharkesh E, Asghari S, and Roshanravan N, Role of Butyrate, a Gut Microbiota Derived Metabolite, in Cardiovascular Diseases: A comprehensive narrative review. Front Pharmacol, 2021. 12:837509.
2. Velsko IM, Chukkapalli SS, Rivera-Kweh MF, Chen H, Zheng D, Bhattacharyya I, Gangula PR, Lucas AR, and Kesavalu L, Fusobacterium nucleatum Alters Atherosclerosis Risk Factors and Enhances Inflammatory Markers with an Atheroprotective Immune Response in ApoE(null) Mice. PLoS One, 2015. 10:e0129795.

Disclosure

J.Q.L. is a part-time employee of GenieBiome Ltd.

Introduction

Abnormalities of mast cells (MCs) structure or function may play prominent roles in irritable bowel syndrome (IBS). While anecdotal evidence suggests that the gut microbiota contributes to dysregulation of MCs, mechanistic insights are limited. We determined that the bacterium Fusobacterium nucleatum (F. nucleatum) played a pathogenic role in IBS via uric acid-mediated MC activation.

Aims & Methods

TNBS/Chronic Restraint Stress was used to establish IBS mouse model. Monocolonization of antibiotic-treated (ABX) mice with F. nucleatum was used to assess its pathogenic role in IBS. Intestinal epithelial cells (IECs) and MCs were used for in vitro experiments. Abdominal withdrawal reflex （AWR） and visceromotor responses (VMR) were used to evaluate visceral hypersensitivity (VHS). The abundance of F. nuclaetum was measured by fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR). The cromolyn sodium (a mast cell stabilizer) was used to determine the role of mast cell. The allopurinol (a uric acid inhibitor) was used to study the role of uric acid. The expression of purine nucleoside phosphorylase (PNP) was measured by qPCR and western blotting (WB).

Results

IBS mouse model showed a higher abundance of F. nucleatum, which was positively corrected with visceral hypersensitivity. And monocolonization of antibiotic-treated (ABX) mice with F. nucleatum induced IBS- like symptoms, including increased VHS and GI transit, by recruiting and activating MCs. F. nucleatum-mediated purine metabolic reprogramming generated uric acid in the intestinal epithelial cells (IECs) that directly stimulated MCs activation via a mechanism involving PNP. This F. nucleatum –driven visceral hypersensitivity and intestinal dysmotility were reversed by mast cell stabilization. Moreover, F. nucleatum-driven MCs recruitment and activation elevated VHS and GI transit was abrogated upon uric acid generation inhibition.

Conclusion

F. nucleatum disrupts gut homeostasis by driving PNP-mediated uric acid production, which activates mast cells and triggers visceral hypersensitivity. This study identifies F. nucleatum-uric acid-mast cell axis as a novel therapeutic target for IBS.

Disclosure

All authors declare no competing interests.

Introduction

Over the world, fecal occult blood testing (FOBT) is used as a colorectal cancer (CRC) screening method for middle age and older age, but there are problems such as sensitivity/specificity issues, low participation rates in primary and secondary screenings, and that the incidence/mortality rates of CRC are still high. Fusobacterium nucleatum (Fn) has been reported to be frequently detected in the feces of CRC patients in recent years, and it has been reported that screening sensitivity can be increased by combining it with Fn PCR testing. However, due to the cost and technical complexity of PCR, it has not yet been widely used in clinical practice. In this study, we developed a simple method using ELISA to measure antibodies against FadA expressed by Fn and evaluated its usefulness.

Aims & Methods

We evaluated FOBT, Fn PCR quantification, and Fn (ELISA) in 41 individuals diagnosed with CRC from April 2020 to December 2022 and 41 healthy volunteers without CRC. Subgroup analysis was also performed in CRC patients at each stage.

Results

The correlation coefficient between Fn PCR quantification and Fn (ELISA) was good at r=0.722, p<0.001. FOBT alone had a sensitivity of 61% and a specificity of 100%. In Fn ELISA, when cut off#1 was set to maximize the AUC in ROC, the sensitivity and specificity were 76% and 78%, respectively, and when combined with FOBT, the sensitivity was 90% and the specificity was 78%. In screening, high specificity is required, so we set cut off#2 Fn ELISA to achieve a specificity of 93% and performed analysis. When combined with FOBT, the sensitivity was 73% and the specificity was 93%. When targeting advanced cancer of stage II or higher, the sensitivity by combining FOBT and Fn ELISA was 100% at cut off#1 and 79% at cut off#2, while for early cancer, the sensitivity was 76% at cut off#1 and 59% at cut off#2, both of which were better than the sensitivity of FOBT alone.

Conclusion

By combining Fn measurement using ELISA with FOBT, it was possible to increase the diagnostic sensitivity and reduce false negatives compared to FOBT alone. There are challenges such as setting appropriate cut-off values for use in screening, but measuring Fn in addition to FOBT may improve screening accuracy and increase the participation rate in secondary screening, and in the future, we would like to evaluate its effectiveness even in populations with low prior probability of screening participation.

Introduction

As the third leading malignancy worldwide, colorectal cancer (CRC) demonstrates strong associations with gut microbiota dysbiosis. Here we investigate, as a key commensal bacterium, how Bifidobacterium breve (B. breve) secreted exopolysaccharides (EPS) modulate anti-tumor immunity through T cell reprogramming.

Aims & Methods

Clinical B. breve abundance was quantified through analysis of patient datasets. Preclinical validation employed three models: 1. CT26 xenografts in NSG vs. BALB/c mice; 2. AOM/DSS-induced colitis-associated carcinoma; 3. EPS bioactivity identification via size-exclusion chromatography and thermal denaturation assays. Immunological effects were analysed through flow cytometry, focusing on stemness-associated markers and exhaustion markers. Systemic immune tracking was mapped across mesenteric lymph nodes, spleen, and tumors.

Results

Metagenomic analysis identified a marked enrichment of Bifidobacterium breve in colorectal cancer (CRC) patients. Subsequent investigation in AOM/DSS-induced CRC murine models demonstrated that oral administration of B. breve substantially attenuated tumor progression. Flow cytometric profiling of tumor infiltrates revealed enhanced T cell stemness and mitigated T cell exhaustion upon bacterial intervention, indicative of functional T cell rejuvenation and altered immunosuppressive microenvironment dynamics. Through systematic fractionation of bacterial supernatants in T cell co-culture systems, we determined that the immunomodulatory activity resides specifically in heat-stable, high-molecular-weight (>3 kDa) components, characteristics aligning with extracellular polysaccharides (EPS). Validation experiments in both NSG immunodeficient and wild-type immunocompetent xenograft models confirmed EPS-mediated antitumor effects require intact immune competence. While oral EPS administration cannot directly transgress the intestinal barrier, comprehensive immune monitoring across mesenteric lymph nodes, splenic tissue, and distal tumor sites revealed coinstantaneous enhancement of T cell stemness profiles, suggesting lymphoid-mediated systemic immunosurveillance potentiation. Notably, EPS supplementation synergized with anti-PD-L1 checkpoint blockade, proposing a microbiota-derived strategy to potentiate immune checkpoint therapy efficacy.

Conclusion

This study elucidates a microbiota-immune circuit where B. breve-EPS promotes T cell stemness through gut lymphoid tissue education, ultimately enhancing systemic anti-tumor responses. Our findings position microbial EPS as novel mucosal checkpoint modulators, suggesting adjuvant use of B. breve polysaccharides could amplify immune checkpoint blockade efficacy.

Introduction

Lipid metabolism dysregulation plays a crucial role in colorectal cancer (CRC) progression. Our perivious study found that upregulation of Slc26a9 promotes colorectal tumorigenesis (Zhang M, et al. Cell Death Discov. 2024). This study investigates the role of Slc26a9 in regulating lipid metabolism through the RRBP1-HSPA5 axis in CRC.

Aims & Methods

In this study, we assessed the expression of Slc26a9 in mouse colorectal cancer tissues using immunohistochemistry and immunofluorescence (IF). Slc26a9-interacting proteins in Colo201 cells, such as RRBP1, were identified through immunoprecipitation followed by mass spectrometry (IP-MS). Bioinformatic analysis of the TCGA and GEO databases was performed to evaluate the correlation between Slc26a9 and RRBP1 expression and their clinical significance. Protein interaction sites were predicted using Protein-Protein Docking. Furthermore, the effects of Slc26a9 overexpression on mitochondrial metabolism, lipid metabolism, the RRBP1-HSPA5 pathway, and endoplasmic reticulum (ER) stress were investigated using Oil Red O staining, Nile Red staining, qRT-PCR, Western blotting (WB), co-immunoprecipitation (co-IP), and IF.

Results

In this study, we employed the widely recognized AOM/DSS protocol to establish a colorectal cancer model in C57BL/6J mice. We observed a significant upregulation of Slc26a9 in colorectal cancer tissues compared to adjacent non-cancerous tissues, consistent with our previous findings in human colorectal cancer samples (Zhang M, et al. Cell Death Discov. 2024). To investigate the precise molecular mechanisms by which Slc26a9 regulates the development of CRC, we performed IP-MS to identify Slc26a9-interacting proteins in Colo201 cells. RRBP1 was found to have the highest binding affinity with Slc26a9 and has been shown to localize at mitochondria–ER contact sites, where it plays a role in regulating organelle interactions. Moreover, GO and KEGG enrichment analyses indicated that Slc26a9 is associated with lipid metabolism in CRC cells. Overexpression of Slc26a9 also promoted endoplasmic reticulum stress, mitochondrial energy metabolism, and lipid accumulation in Colo201 cells. Moreover, Slc26a9 enhanced the expression of markers related to endoplasmic reticulum stress (PERK-ATF4, IRE1α-XBP1, and ATF6), mitochondrial metabolism (Mitofilin, MFN1, and COXIV), and lipid metabolism (FASN, ACC, and SCD1). Moreover, Slc26a9 upregulation also enhanced the expression of the endoplasmic reticulum stress–related signaling pathway RRBP1/HSPA5. Protein-protein docking analysis revealed the formation of multiple hydrogen bonds between Slc26a9 and RRBP1, indicating a high degree of structural complementarity and strong binding affinity between the two proteins. Co-IP and IF experiments demonstrated that Slc26a9 interacts with RRBP1 and co-localizes with it. In addition, bioinformatics analysis of GEO dataset (GSE39582) showed a positive correlation between Slc26a9 and RRBP1 expression in human CRC tissues, with their co-high expression predicting poor overall survival.

Conclusion

These findings highlight that Slc26a9 interacts with RRBP1 to activate the HSPA5-mediated ER stress response, driving mitochondrial metabolism-lipid metabolic reprogramming and promoting CRC progression. Slc26a9 and RRBP1 may serve as potential prognostic biomarkers, and their axis represents a novel therapeutic target for CRC.