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. 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. 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. 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.

BACKGROUND:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths, primarily due to late-stage diagnosis. In this multicenter study, our goal is to identify functional biomarkers that stratify the risk of HCC in patients with cirrhosis (CP) for early diagnosis. METHODS:Five thousand and eight serum proteins (Somascan) were analysed in Cohort A (477 CP, including 125 HCC). Clustering analysis of the TGF-β pathway-associated protein signature was performed in a longitudinal, prospective Cohort B (312 CP, in which 18 cases developed HCC over a 5-year follow-up period). Next, a multivariable prediction model was built using logistic regression analysis of cross-sectional data from a matched subgroup (n = 328, Cohort C). Model performance was 10-fold cross-validated across the entire Cohort A (n = 477). RESULTS:Longitudinal follow-up analysis revealed that patients with elevated TGF-β-related protein signature displayed a five-fold increased risk of developing HCC (9.68% vs. 1.91%). Compared to cirrhosis, serum MSTN, TGFBR2, and AFP levels raised in HCC were validated by ELISA (n = 200, odds ratio = 1.4-2.9, p < 0.05). In Cohort C, 88 proteins were significantly altered in HCC compared to cirrhosis (p < 0.05). The six-protein panel (TGFBR2, MSTN, AFP, COL18A1, GLUL, TP63) displayed a strong performance in the matched cohort C (AUC 0.87, sensitivity 0.88, specificity 0.72), alongside four clinical factors (Age, Sex, BMI, Bilirubin). A 10-fold cross-validation demonstrated a mean AUC of 0.86 in cohort A, with strong predictive power in obese/MASLD/ALD-related patients (AUCs: 0.862-0.921). CONCLUSIONS:The mechanism-based panel effectively stratifies HCC risk in cirrhotic patients, underscoring the need for Phase II/III validation.

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.

Obesity and fatty liver diseases-metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH)-affect over one-third of the global population and are exacerbated in individuals with reduced functional aldehyde dehydrogenase 2 (ALDH2), observed in approximately 560 million people. Current treatment to prevent disease progression to cancer remains inadequate, requiring innovative approaches. We observe that Aldh2-'-- '- and Aldh2-'-Sptbn1+'-- '- Sptbn1 + '- mice develop phenotypes of human metabolic syndrome (MetS) and MASH with accumulation of endogenous aldehydes such as 4-hydroxynonenal (4-HNE). Mechanistic studies demonstrate aberrant transforming growth factor b (TGF-b) signaling through 4-HNE modification of the SMAD3 adaptor SPTBN1 (b2-spectrin) to pro- fibrotic and pro-oncogenic phenotypes, which is restored to normal SMAD3 signaling by targeting SPTBN1 with small interfering RNA (siRNA). Significantly, therapeutic inhibition of SPTBN1 blocks MASH and fibrosis in a human model and, additionally, improves glucose handling in Aldh2-'-- '- and Aldh2-'-Sptbn1+'-- '- Sptbn1 + '- mice. This study identifies SPTBN1 as a critical regulator of the functional phenotype of toxic aldehyde-induced MASH and a potential therapeutic target.

Supplementary Materials and Methods

Supplementary Figures and legends

Dysregulated transforming growth factor‐beta (TGF‐β) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF‐β signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild‐type mice, high‐fat diet‐induced fatty liver is worse in TGF‐β signaling‐deficient mice (SPTBN1+/−) and the mutant mice had reduced SIRT6 abundance in the liver. Therefore, we hypothesized that altered reciprocal regulation between TGF‐β signaling and SIRT6 contributes to these liver pathologies. We found that deficiency in SMAD3 or SPTBN1 reduced SIRT6 mRNA and protein abundance and impaired TGF‐β induction of SIRT6 transcripts, and that SMAD3 bound to the SIRT6 promoter, suggesting that an SMAD3‐SPTBN1 pathway mediated the induction of SIRT6 in response to TGF‐β. Overexpression of SIRT6 in HCC cells reduced the expression of TGF‐β‐induced genes, consistent with the suppressive role of SIRT6 on TGF‐β signaling. Manipulation of SIRT6 abundance in HCC cells altered sterol regulatory element‐binding protein (SREBP) activity and overexpression of SIRT6 reduced the amount of acetylated SPTBN1 and the abundance of both SMAD3 and SPTBN1. Furthermore, induction of SREBP target genes in response to SIRT6 overexpression was impaired in SPTBN1 heterozygous cells. Thus, we identified a regulatory loop between SIRT6 and SPTBN1 that represents a potential mechanism for susceptibility to fatty liver in the presence of dysfunctional TGF‐β signaling.

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that β2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)–binding protein (SREBP)–stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.

Inactivation of the transforming growth factor-β (TGF-β) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/− and elf+/−/Smad4+/− mutant mice. We found that embryonic liver fodrin (ELF), a β-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell–cell contacts and E-cadherin-β-catenin-dependent epithelial cell–cell adhesion is disrupted in elf+/−/Smad4+/− mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-β stimulation. In contrast, elf+/−/Smad4+/− mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-β signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.

Abstract RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFβ/SMAD signaling. SMAD3 and its adaptors, such as β2SP, are important mediators of TGFβ signaling and regulate gene expression to suppress stem cell–like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/β2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3+/−), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFβ1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients. Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFβ in liver carcinogenesis.