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.
