Metabolomics-based and functional validation to explore the effect of the gut microbe-associated metabolite guanidinoacetic acid on rectal adenocarcinoma

The gut microbiota plays a crucial role in the pathogenesis of rectal adenocarcinoma (READ). While studies have established associations between microbial alterations and READ, the dynamics of gut microbiota-host interactions within this specific disease context remain to be fully elucidated. This study aimed to characterize the structural landscape of the gut microbiota and the profile of faecal metabolites in patients with READ. Faecal samples were collected from 33 individuals with READ and 34 healthy controls (HC), with post-neoadjuvant treatment faecal samples also collected from the 5 READ patients, and 16 S rRNA gene amplification sequencing and untargeted liquid chromatography-mass spectrometry metabolomics analysis were performed. We employed methods such as weighted correlation network analysis (WGCNA) and molecular docking to identify target gene-related information, and performed in vitro experiments to explore the effects of guanidinoacetic acid (GAA) on colorectal cancer cell lines. Bacillota (formerly Firmicutes), Pseudomonadota (formerly Proteobacteria), Actinomycetota (formerly Actinobacteria), and Bacteroidota (formerly Bacteroidetes) were the most abundant phyla in patients with READ and healthy controls (HC). At the genus level, Peptostreptococcus exhibited marked enrichment in the READ cohorts, whereas Faecalibacterium was dominant in the HC group. The predicted microbial functional analysis indicated a significant increase in the metabolism of betaine, guanidinoacetic acid (GAA), L-tryptophan, arachidonic acid, and arachidic acid. Moreover, by comparing the faecal metabolites between the HC and READ groups, Pearson's correlation analysis demonstrated significant interactions between six microbiota taxa and nine metabolites, suggesting specific human metabolic pathways. The cellular function results demonstrated that GAA promoted the proliferation, invasion, and migration of colorectal cells while inhibiting apoptosis. Further analysis revealed that GAA may promote CRC progression of colorectal cancer by affecting molecules such as KLB, CA2, CSTG, CYP4F12, and GZBM. Through integrated analysis, this study links gut microbial metabolites to READ and identifies GAA as a metabolite with possible relevance to the disease process, indicating a direction for further validation.