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Microbial metabolite ammonia disrupts CEACAM1-TGF-β signaling to promote colon cancer

ABSTRACT: Colorectal cancer (CRC) incidence is rising globally and anticipated to become the leading cause of cancer death in younger individuals. Potential risk factors are diet induced obesity and altered microbiomes that lead to accumulation of toxic metabolite accumulation. However, how ammonia and other microbial metabolites impact key signaling pathways, such as TGF-β signaling, 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. We found that in an obesity induced mouse model of cancer, altered microbial populations and ammonia promote Caspase-3-mediated cleavage of SMAD3 adaptor βII-spectrin (SPTBN1). Cleaved SPTBN1 fragments form adducts with ammonia to induce pro-inflammatory cytokine expression and alter TGF-β signaling driving CRC. Extending on Alphafold docking simulations, we identified that ammonia interacts with six polar residues at SPTBN1 (S553, Y556, S663, Y666, N986, and T1178) of cleaved SPTBN1 fragments 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 restore TGF-β signaling by reducing the abundance of SPTBN1 cleaved fragments. Importantly, SPTBN1 siRNA blocks ammonia toxicity and restore normal TGF-β signaling in CRC cells. Moreover, 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, with both molecules playing pivotal roles in CRC progression. Our study identifies mechanistic insights into how microbial metabolites target TGF-β a major signaling pathways to promote CRC.

ORGANISM(S): Homo sapiens

PROVIDER: GSE281613 | GEO | 2024/11/16

REPOSITORIES: GEO

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Project description:Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at the various developmental stages before, during, and after palate fusion using GeneChip? microarrays. Ceacam1 was one of the highly up-regulated genes during and after fusion in palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was expressed at a very low level in palatal epithelium before fusion, but highly expressed in the midline of the palate during and after fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1-/-) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1-/- mice. TGF?3 expression, apoptosis, and cell proliferation in palatal epithelium were not effected in the palate of Ceacam1-/-mice. CEACAM1 expression was down-regulated in Tgfb3-/- palate. However, exogenous TGF?3 did not induce CEACAM1 expression. These results suggest that CEACAM1 has roles in both the initiation of palate formation via epithelial cell adhesion and TGF signaling has some indirect effect on CEACAM1. Global gene expression profiling of palatal processes before, during and after fusion of palatal shelves We used microarray to investigate the gene expression of palatal tissue during palatal development. Palatal processes were microdissected at the stages of palatal development (before, during and after fusion) for RNA extraction and hybridization on Affymetrix microarrays.

Project description:Objective We have previously shown that expression of the CEACAM1 long isoform (CC1-L) in metastatic colorectal cancer (CRC) cells results in significant inhibition of liver metastasis via Chemokine (C-C motif) Ligand 2 (CCL2) and Signal Transducer and Activator of Transcription 3 (STAT3) signaling. Yet, despite recent advances in identifying molecules involved in the CC1-L inhibition of metastasis, much remains to be elucidated regarding the molecular mechanisms orchestrating this pathway. Design We performed an untargeted transcript profiling and a phosphokinase screen between CC1-L-expressing and non-expressing (CT) CRC cells. Identified targets were validated in vitro and in vivo for their involvement in signaling and invasion or metastasis. We validated our conclusions in CRC patient cohorts for time to metastasis development and long-term survival. Results Untargeted transcript profiling revealed high Decorin (Dcn) expression in CC1-L-expressing cells versus CT cells. Decorin was detected at the peri-endothelial layers of large blood vessels and in liver stellate cells. In metastatic lesions, it was expressed in pericyte-like cells covering capillary endothelium. Phosphokinase differential screening revealed reduced Ephrin type-A receptor 2 (EphA2) expression and activity in CC1-L- versus CT-expressing cells. Treatment of CT cells with recombinant Dcn led to decreased migration and invasion and decreased EphA2-mediated Erk and Akt signaling. CRC patients exhibiting high CC1 and DCN expression, in combination with low EPHA2 expression, benefit from longer 10-year survival than those with high EPHA2 expression. Conclusion CC1-L expression in poorly differentiated CRC inhibits liver metastasis through increased Decorin expression and EphA2-mediated signaling. Quadriplicate samples of each mouse colon cancer cell lines (control cells versus those expressing either short or long isoform of CEACAM1) have been used for RNA extraction

Project description:Most available CRC cell lines have lost their TGF-beta response through the acquisition of mutations either in TGF-beta type II receptor (TGFBR2) or the intracellular mediator SMAD4 (Reviewed in Markowitz et al. 2002). To study the effect of TGF-beta in CRC epithelial cells, we restored wild-type TGFBR2 expression in the CRC cell line LS174T (LS). Ls174T cells are mutant for beta-catenin and therefore exhibit constitutive Wnt activity, which results in the expression of a genetic profile similar to that of intestinal epithelial progenitor cells (van de Wetering, Sancho et al. 2002). They thus reflect tumor cells at the initial stages of tumorogenesis. In addition, Ls174T cells bear mutations in the TGF-beta receptor II (TGFBR2) sequence at the BATRII locus (Grady and Markowitz 2002). This mutation gives rise to a non-functional truncated form of the receptor lacking the transmembrane domain and the serine-threonine kinase domain (Parsons, Myeroff et al. 1995). We generated inducible clones of Ls174T cells, where the expression of TGFBR2 could be controlled at will by the presence or absence of doxycycline (LSTGFBR2 cell line). Re-expression of wild-type receptor in Ls174T cells (LSTGFBR2) restored TGF-beta signalling resulting in strong phosphorylation of SMADs and activation of SMAD-binding reporter activity. Functionally, TGF-beta signalling in LSTGFBR2 resulted in a strong cytostatic response. By genome wide expression profiling using microarrays we investigated genes regulated by TGF-beta in this cell line. LsTGFBR2 inducible cells were generated by the Invitrogen T-Rex system following manufacturers instructions. LSTGFBR2 were seeded at 60% confluence in the presence or absence of Doxycycline and treated with TGF-M-NM-21 for 16 hours. Gene expression profiles were measured in duplicate using HG-U133 plus 2.0. We used RMA background correction, quantile normalization and RMA summarization (Gautier et al., 2004). A TGF-M-NM-2 response signature was obtained by selecting genes with limma P-value < 0.05 and at least two fold up-regulation in TGF-M-NM-2 treated LSTGFBR2 cells.

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Microbial metabolite ammonia disrupts CEACAM1-TGF-β signaling to promote colon cancer

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