Abstract 407: Loss of Smad3 alters host-microbial interactions, predisposing the colonic epithelium to inflammation

Abstract

Abstract Introduction: The TGFβ pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Smad3−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 signaling and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 signaling on colonic bacterial composition we used the Smad3−/− GEM. We designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on cecal DNA extracted from Smad3−/-− and Smad3+/+ mice. To further examine the host-microbial interaction we extracted RNA and protein from the cecum of Smad3−/− and Smad3+/+ mice and ran Real Time-PCR or Western Blot for different Toll-like Receptor Pathway components. Results: qRT-PCR showed that Smad3−/-− mice have a significantly lower quantity of Bacteroides sp; the introduction of Helicobacter hepaticus results in a significant increase in B. distasonis and a significant decrease in B. thetaiotaomicron in Smad3−/− mice. These data suggest that loss of SMAD3 and the addition of Helicobacter hepaticus alters the colonic microflora. Previous studies illustrated the importance of epithelial signaling in microbial induced inflammation. To examine if Smad3 is altering the Toll-like receptor pathway we examined RT-PCR of the cecum and found a significant increase in Tlr4, Irak4, Cd14, Myd88, Nfkb, Cox2, and Nos2 mRNA in the Smad3−/−− mice compared to Smad3+/+ mice. Western blots confirmed these data by showing that cecal tissue from Smad3−/− mice has increased IRAK4 and pNF-κB. Conclusion: Smad3−/− mice show heightened Toll-like receptor pathway activity which leads to a hyper-inflammatory response of the mucosa to endogenous microbes. Further, loss of SMAD3 results in dysbiosis of the gut microbiota. These findings suggest that SMAD3 plays a role in microbial-host homeostasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 407. doi:1538-7445.AM2012-407