The distinct roles of Negativicoccus and Fusobacterium in proximal- and late-onset colorectal cancer

Despite the emerging role of the gut microbiome in colorectal cancer (CRC), its significance in early-onset CRC (EOCRC, n = 76) and stool samples (n = 33) by 16S rRNA sequencing. Diversity indices were compared using Wilcoxon tests, compositional differences assessed by PERMANOVA, and correlations were performed in paired samples. Correlation analysis revealed strong associations between microbial phyla (Bacteroidetes, R = 0.86, p = 4.7 × 10⁻⁶; Firmicutes, R = 0.65, p = 3.4 × 10⁻³; Verrucomicrobiota, R = 0.81, p = 4.8 × 10⁻⁵; and Fusobacterium, R = 0.80, p = 7.3 × 10⁻⁵) and major genera (Bacteroides, R = 0.88, p = 1.7 × 10⁻⁵; Fusobacetrium, R = 0.75, p = 1.5 × 10⁻³; Blautia, R = 0.77, p = 8.5 × 10⁻⁴; and Bifidobacterium, R = 0.81, p = 3.3 × 10⁻⁴) across sample types, validating the use of anal swabs. However, Actinobacteriota and Prevotella were not correlated, likely reflecting the perianal skin-associated microbiota and underscoring the need for validation against stool or mucosal biopsies. Importantly, anal swabs revealed associations between Negativicoccus and proximal CRC (p = 0.047) and between the Fusobacteriota phylum and LOCRC (p = 0.042), suggesting subtype-specific CRC subtypes. In mechanistic studies, using the mucous-secreting HT-29 MTX cell line, we observed that Negativicoccus was associated with activation the RAS/MAPK pathway, upregulated c-MYC, KRAS, MAPK1, and Cyclin D1 (p p Fusobacterium modulates the WNT/β-catenin pathway, increasing β-catenin and AXIN1 (p p Negativicoccus influencing proliferation and inflammation, whereas Fusobacterium promotes migration and invasion. Understanding these pathways offers potential for harnessing the gut microbiome's diagnostic and therapeutic power in CRC.