Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

Aortic aneurysm is predominantly found in the ascending aorta in patients with Marfan syndrome (MFS). However, descending aortic disease has emerged as a problem since people are living longer because of improved medical and surgical management of the ascending aorta. Diagnostic procedures before disease onset and the mechanisms involved in the transition of normal aortic tissue to aneurysm remain unclear. We determined signs of descending aortic disease before disease onset in mice with a mutation in the fibrillin 1 gene (Fbn1(+/C1039G)), a validated mouse model of disease susceptibility and progression of aortic aneurysm of MFS. We analyzed a tubular unfixed non-aneurysmal descending thoracic aorta from 8-month-old wild-type and Fbn1(+/C1039G) mice by a tubular biaxial tester that works in conjunction with a two-photon nonlinear microscope. Fbn1(+/C1039G) mouse aorta was more compliant in the circumferential direction. Two-photon imaging showed defective organization of adventitial collagen fibers in the pressurized aortas of Fbn1(+/C1039G) mice. Moreover, disruption in the elastic lamina was noted in the absence of aneurysms in pressurized aortas but not unpressurized aortas of Fbn1(+/C1039G) mice. At the molecular level, this altered tissue behavior in non-aneurysmal descending aortas of Fbn1(+/C1039G) mice was accompanied by an increasing trend of canonical but not noncanonical, transforming growth factor-β (TGFβ) signaling. Finally, assays of in vitro collagen lattice formation in mouse wild-type and TGFβ1-deficient embryonic fibroblasts indicate that TGFβ1 can regulate collagen organization. The ability to reveal the presence of altered biomechanics and microstructure coupled with subtle changes in TGFβ signaling provides a novel surrogate measure of tissue susceptibility to aneurysm before disease onset.

Introduction: The TGFB 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 Tgfb1−/−Rag2−/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 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 on colonic bacterial composition we used GEM models, Tgfb1−/−Rag2−/− and Tgfb1+/+Rag2−/−. The cecal microflora of 10 Tgfb1−/−Rag2−/− and 10 Tgfb1+/+Rag2−/− mice were isolated and serially diluted onto brucella (BRU), bacteroides bile esculin (BBE), and laked kanomycin-vancomycin (LKV) media under anaerobic conditions. Colony forming units (CFUs) were enumerated. Individual colony types were then streaked onto trypitcase soy agar with 5% sheep blood and grown anaerobically and aerobically. The bacteria were then gram-stained and biotyped utilizing a Dade Berhing MicroScan instrument. To further examine the bacterial composition 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 fecal DNA. Results: The Dade Berhing Instrument showed that Tgfb1−/−Rag2−/− mice had a 4 fold increase in bacterial load and a 28 fold increase in Bacteroides species when compared with Tgfb1+/+Rag2−/− mice. The qRT-PCR results showed an increase in Bacteroides fragilis and Bacteroides distasonis and a significant decrease in Bacteroides thetaiotaomicron in the Tgfb1−/−Rag2−/− mice. These data suggest that loss of TGFβ1 alters the colonic microflora. Previous studies illustrated the importance of bacterial nutrient sources on bacterial composition. To examine if TGFβ1 is altering nutrient availability in the colon a previous micro-array was analyzed for candidate genes associated with glycoprotein metabolism. This showed changes in fucose metabolizing enzymes with the loss of TGFβ1. Conclusion: These findings suggest that TGFβ1 plays a role in bacterial load maintenance, possibly by altering available nutrient sources and when disrupted, can cause abnormalities in pathobionts (commensal bacterial with pathogenic potential) which could then lead to increased inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1957.

Introduction: TGFβ plays important role in T cell development, differentiation, tolerance and autoimmune diseases. However, recent studies have shown an important role for TGF-β1 and β3 in pathogenic helper T-cell (Th17) differentiation in the presence of IL-1β, IL-6 and IL-23. SMAD3 is mediates TGFβ signaling in many cell types including lymphocytes. Smad3 /- mice develop inflammation-induced colon cancer upon Helicobacter hepaticus infection suggesting that TGFβ1 signaling through SMAD3 is essential to prevent inflammation and progression to colon cancer. In this study we investigated the role of SMAD3 in inflammation using mice deficient in Smad3. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines are determined by ELISA, CBA and MSD methods. Cells are stained with antibodies for CD4, CD25, CD62L and intracellular FOXP3 and Helios. Results: Smad3-/- mice exhibit inflammatory response upon infection with commensal bacterium H. hepaticus. We find that while SMAD3 signaling is important for inducible Treg cell generation and for their maintenance, deficiency of SMAD3 increases IL-6 and IL-17 levels suggesting a regulatory role for SMAD3 in Th17 cell differentiation. Conclusions: While SMAD3 signaling is important for iTreg cell generation and maintenance, it is not required for Th17 cell differentiation. In addition, we find that SMAD3 signaling negatively regulates Th17 differentiation through induction of FOXP3.

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