SMAD4 Mutation Drives Gut Microbiome Shifts Toward Tumor Progression in Colorectal Cancer

Colorectal cancer (CRC) progression is driven by a series of sequential mutations in key driver genes, yet the factors underpinning tumor advancement and metastasis remain incompletely understood. Mutations in TP53 and SMAD4, in particular, are associated with poor treatment response and enhanced CRC pathogenesis. Although gut microbiome dysbiosis is implicated in CRC initiation and inflammation, the interactions between the microbiome and specific CRC driver mutations, especially those promoting metastasis, are poorly defined. In this study, we used triple mutant (Apc, Kras, Tp53; AKP) and quadruple mutant (Apc, Kras, Tp53, Smad4; AKPS) organoid-based orthotopic mouse models of CRC to examine the impact of SMAD4 mutation on tumor progression, metastasis, and microbiome composition. AKP and AKPS organoids were endoscopically implanted into the colons of individually housed C57BL/6 mice, and fecal samples were collected weekly over an 8-week period. Our results reveal significant differences in metastatic potential and microbial community dynamics between the two tumor models. AKPS tumors exhibited metastasis to the lymph nodes, liver, and lungs, whereas AKP tumors remained confined to the colon. Longitudinal microbiome analysis showed shifts in microbial composition within each tumor model. Both AKP and AKPS models demonstrated enrichment of Faecalibaculum and a decrease in Dubosiella over time; however, additional shifts were observed with distinct taxa associated with late-stage tumors in each group. Notably, the AKPS model exhibited higher relative abundances of pro-inflammatory taxa, including Turicibacter, Romboutsia, and Akkermansia , suggesting that SMAD4 mutation promotes a more immunosuppressive and pro-metastatic microbiome profile. These findings underscore the role of SMAD4 in modulating the microbiome in a manner that favors CRC metastasis and suggests potential microbial targets for therapeutic intervention to slow CRC progression. This work provides new insights into the microbiome’s role in CRC mutagenesis and metastasis, highlighting the interplay between host genetics and gut microbiota in driving cancer aggressiveness.