Abstract 6533: Microbial metabolite ammonia disrupts CEACAM1 TGF-β signaling to drive colon cancer progression

Abstract Background: Colorectal cancer (CRC) is rising alarmingly in younger populations, potentially arising from additional risk factors such as obesity, pro-inflammatory microbiomes and 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 TGF-β signaling pathway, to drive CRC progression. Methods: Through human TCGA-CRC data analyses, with animal model studies, molecular docking and signaling studies, we examine how gut microbiome alterations, particularly ammonia production, modulate key oncogenic pathways involving Smad3 adaptor, SPTBN1, CEACAM1, and TGF-β. Our work builds on two of our recent publications (Cell Rep.2024.43(9):114676 and Sci Transl Med.2021.13(624): eabk2267). Results We observe 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 pro-inflammatory cytokine expression and disrupt TGF-β signaling. Extending on AlphaFold docking simulations, we identified that ammonia interacts with six polar residues at SPTBN1 (S553, Y556, S663, Y666, N986, and T1178) to form hydrogen bonds that disrupt downstream SMAD3 signaling, altering TGF-β signaling to a protumor genic 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 HCT116 (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 (ITIM) of CEACAM1 cytoplasmic domain, identifying a potential axis that is harnessed by the altered microbiome. Conclusions: Our study identifies mechanistic insights into how microbial metabolites such as ammonia target TGF-β, a major signaling pathway, to promote CRC. These findings underscore the therapeutic potential of targeting SPTBN1 to restore tumor-suppressive TGF-β signaling and improve outcomes for CRC patients. Citation Format: Krishanu Bhowmick, Puja Ghosh, Xiyan Xiang, Xiaochun Yang, Taj Mohammad, Bibhuti Mishra, Md. Imtaiyaz Hassan, Adrian R Krainer, Srinivasan Dasarathy, Keith A Crandall, Lopa Mishra. Microbial metabolite ammonia disrupts CEACAM1 TGF-β signaling to drive colon cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6533.