Microbial metabolite ammonia disrupts TGF-β signaling to promote colon cancer

Colorectal cancer (CRC) is rising alarmingly in younger populations, potentially arising from factors, such as obesity, proinflammatory gut microbiome, and the accumulation of toxic metabolites. However, how metabolites such as ammonia impact key signaling pathways to promote CRC remains unclear. Our study investigates a critical link between gut microbiome alterations, ammonia, and their toxic effects on the transforming growth factor beta (TGF-β) signaling pathway, driving CRC progression. We observed altered microbial populations in an obesity-induced mouse model of cancer, where ammonia promotes caspase-3-mediated cleavage of the SMAD3 adaptor βII-spectrin (SPTBN1). Cleaved SPTBN1 fragments form adducts with ammonia that induce proinflammatory cytokine expression and disrupt TGF-β signaling. Extending from AlphaFold docking simulations, we identified that ammonia interacts with N-terminal SPTBN1 potentially through residues D81, Y556, S663, Y666, N986, and D1177 to form hydrogen bonds that disrupt downstream SMAD3 signaling, altering TGF-β signaling to a protumorigenic phenotype. Blocking SPTBN1, through an SPTBN1-specific siRNA, blocks ammonia toxicity and restores normal SMAD3/TGF-β signaling by reducing the abundance of SPTBN1-cleaved fragments in SW480 and Caco-2 (CRC) cell lines. In addition, our research establishes crosstalk between TGF-β signaling and a microbial sensor, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is significantly overexpressed in CRC patients. We identified CEACAM1-SPTBN1 interactions at specific residues (E517 and Y520) within the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 cytoplasmic domain, identifying a potential axis that is harnessed by the altered microbiome. Our study identifies mechanistic insights into how microbial metabolites target TGF-β as a major signaling pathway to promote CRC.