FAECALIBACTERIUM PRAUSNITZII SUPPRESSES COLORECTAL TUMORIGENESIS BY PRODUCING HYPOXANTHINE AND INHIBITING FUSOBACTERIUM NUCLEATUM

Background and Aim: Whether pathogenic bacteria promoting colorectal cancer (CRC), such as Fusobacterium nucleatum (Fn), can be inhibited by specific beneficial bacteria and the mechanisms of action have not been investigated. This study identified a potential probiotic Faecalibacterium prausnitzii (Fp) that played inhibitory effects on CRC and Fn and fully elucidated its functional mechanism.
Methods: Metagenomic data from 589 Chinese subjects was analyzed. Metabolites of Fp was identified by non-targeted LC-MS. Colon cancer cells (HT-29, HCT116 and Caco-2) were co-cultured with Fn for 4h, then treated with Fp or E. coli (Ec) supernatant, Hypoxanthine (Hx), or broth control after removal of Fn. Gene expression profiles were analyzed by RNA sequencing. C57BL/6J-Apc^Min/+ mice were administered Fp, Ec, Hx, broth, Fn+Fp, or Fn+Ec/broth to evaluate their effects on colon tumor development. IHC was conducted to assess Ki-67⁺ proliferative cells and Nt5e expression in colon tissues of mice.
Results: Metagenomics identified Fp to be significantly decreased in CRC patients and the number one bacterium inversely correlated with Fn. Fp significantly inhibited the growth, clonogenicity, and cell cycle progression of colon cancer cells and drove them to apoptosis. Meanwhile, Fp significantly inhibited the growth and aggregation of Fn. Moreover, Fn significantly promoted colon cancer cell growth and migration, and these promoting effects were abolished by subsequent Fp treatment. Fp’s effective component(s) was non-protein by heat and protease K treatments. Metabolomics identified the purine derivative Hx as the top metabolite of Fp. Hx significantly inhibited the growth of colon cancer cells and Fn and also diminished the growth-promoting effect of Fn in vitro. Fp and Hx treatments both significantly reduced colon tumor load and proliferative colonic cells compared with controls in mice. Fn promoted colon tumor formation as expected, while the accompanying Fp treatment (Fn+Fp) significantly decreased tumor number, tumor load, fecal Fn abundance, and proliferative colonic cells versus Fn+Ec/broth. Transcriptome analysis identified oncogenic genes to be upregulated by Fn but downregulated by subsequent Fp treatment, including NT5E, which participates in purine salvage pathway and promotes cancer development. Western blot showed that NT5E was upregulated by Fn co-culture but downregulated by subsequent Fp or Hx treatments in vitro. NT5E knockdown significantly inhibited cell growth and migration and diminished Hx’s inhibitory effect, indicating Hx functions largely through downregulating NT5E. Moreover, Fp and Hx treatments also significantly downregulated Nt5e in mouse colon tissues compared with controls.
Conclusions: Fp suppresses CRC development directly or through antagonizing the tumor-promoting effect of Fn by producing Hx, which functions by inhibiting NT5E.