Amber Bradley

Dietary selenium deficiency exacerbates DSS-induced epithelial injury and AOM/DSS-induced tumorigenesis.

Selenium (Se) is an essential micronutrient that exerts its functions via selenoproteins. Little is known about the role of Se in inflammatory bowel disease (IBD). Epidemiological studies have inversely correlated nutritional Se status with IBD severity and colon cancer risk. Moreover, molecular studies have revealed that Se deficiency activates WNT signaling, a pathway essential to intestinal stem cell programs and pivotal to injury recovery processes in IBD that is also activated in inflammatory neoplastic transformation. In order to better understand the role of Se in epithelial injury and tumorigenesis resulting from inflammatory stimuli, we examined colonic phenotypes in Se-deficient or -sufficient mice in response to dextran sodium sulfate (DSS)-induced colitis, and azoxymethane (AOM) followed by cyclical administration of DSS, respectively. In response to DSS alone, Se-deficient mice demonstrated increased morbidity, weight loss, stool scores, and colonic injury with a concomitant increase in DNA damage and increases in inflammation-related cytokines. As there was an increase in DNA damage as well as expression of several EGF and TGF-β pathway genes in response to inflammatory injury, we sought to determine if tumorigenesis was altered in the setting of inflammatory carcinogenesis. Se-deficient mice subjected to AOM/DSS treatment to model colitis-associated cancer (CAC) had increased tumor number, though not size, as well as increased incidence of high grade dysplasia. This increase in tumor initiation was likely due to a general increase in colonic DNA damage, as increased 8-OHdG staining was seen in Se-deficient tumors and adjacent, non-tumor mucosa. Taken together, our results indicate that Se deficiency worsens experimental colitis and promotes tumor development and progression in inflammatory carcinogenesis.

Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage

Patients with inflammatory bowel disease are at increased risk for colon cancer due to augmented oxidative stress. These patients also have compromised antioxidant defenses as the result of nutritional deficiencies. The micronutrient selenium is essential for selenoprotein production and is transported from the liver to target tissues via selenoprotein P (SEPP1). Target tissues also produce SEPP1, which is thought to possess an endogenous antioxidant function. Here, we have shown that mice with Sepp1 haploinsufficiency or mutations that disrupt either the selenium transport or the enzymatic domain of SEPP1 exhibit increased colitis-associated carcinogenesis as the result of increased genomic instability and promotion of a protumorigenic microenvironment. Reduced SEPP1 function markedly increased M2-polarized macrophages, indicating a role for SEPP1 in macrophage polarization and immune function. Furthermore, compared with partial loss, complete loss of SEPP1 substantially reduced tumor burden, in part due to increased apoptosis. Using intestinal organoid cultures, we found that, compared with those from WT animals, Sepp1-null cultures display increased stem cell characteristics that are coupled with increased ROS production, DNA damage, proliferation, decreased cell survival, and modulation of WNT signaling in response to H2O2-mediated oxidative stress. Together, these data demonstrate that SEPP1 influences inflammatory tumorigenesis by affecting genomic stability, the inflammatory microenvironment, and epithelial stem cell functions.

MTG16 contributes to colonic epithelial integrity in experimental colitis

Objective The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8−/− and Mtgr1−/− mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. Methods Baseline and stress induced colonic phenotypes were examined in Mtg16−/− mice. To unmask phenotypes, we treated Mtg16−/− mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. Results Mtg16−/− mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16−/− mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1+, F4/80+, CD11c+ and MHCII+; CD11c+) and Th1 adaptive (CD4) immune cells in Mtg16−/− colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16−/− colons. Compared with wild-type mice, Mtg16−/− mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16−/− recipients did not rescue the Mtg16−/− injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16−/− mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16−/− mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. Conclusions These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.

Structure of pyrazole derivatives impact their affinity, stoichiometry, and cooperative interactions for CYP2E1 complexes.

CYP2E1 plays a critical role in detoxification and carcinogenic activation of drugs, pollutants, and dietary compounds; however, these metabolic processes can involve poorly characterized cooperative interactions that compromise the ability to understand and predict CYP2E1 metabolism. Herein, we employed an array of ten azoles with an emphasis on pyrazoles to establish the selectivity of catalytic and cooperative CYP2E1 sites through binding and catalytic studies. Spectral binding studies for monocyclic azoles suggested two binding events, while bicyclic azoles suggested one. Pyrazole had moderate affinity toward the CYP2E1 catalytic site that improved when a methyl group was introduced at either position 3 or 4. The presence of methyl groups simultaneously at positions 3 and 5 blocked binding, and a phenyl group at position 3 did not improve binding affinity. In contrast, pyrazole fusion to a benzene or cyclohexane ring greatly increased affinity. The consequences of these binding events on CYP2E1 catalysis were studied through inhibition studies with 4-nitrophenol, a substrate known to bind both sites. Most pyrazoles shared a common mixed cooperative inhibition mechanism in which pyrazole binding rescued CYP2E1 from substrate inhibition. Overall, inhibitor affinities toward the CYP2E1 catalytic site were similar to those reported in binding studies, and the same trend was observed for binding at the cooperative site. Taken together, these studies identified key structural determinants in the affinity and stoichiometry of azole interactions with CYP2E1 and consequences on catalysis that further advance an understanding of the relationship between structure and function for this enzyme.

Myeloid translocation genes differentially regulate colorectal cancer programs

Myeloid translocation genes (MTGs), originally identified as chromosomal translocations in acute myelogenous leukemia, are transcriptional corepressors that regulate hematopoietic stem cell programs. Analysis of The Cancer Genome Atlas (TCGA) database revealed that MTGs were mutated in epithelial malignancy and suggested that loss of function might promote tumorigenesis. Genetic deletion of MTGR1 and MTG16 in the mouse has revealed unexpected and unique roles within the intestinal epithelium. Mtgr1−/− mice have progressive depletion of all intestinal secretory cells, and Mtg16−/− mice have a decrease in goblet cells. Furthermore, both Mtgr1−/− and Mtg16−/− mice have increased intestinal epithelial cell proliferation. We thus hypothesized that loss of MTGR1 or MTG16 would modify Apc1638/+-dependent intestinal tumorigenesis. Mtgr1−/− mice, but not Mtg16−/− mice, had a 10-fold increase in tumor multiplicity. This was associated with more advanced dysplasia, including progression to invasive adenocarcinoma, and augmented intratumoral proliferation. Analysis of chromatin immunoprecipitation sequencing data sets for MTGR1 and MTG16 targets indicated that MTGR1 can regulate Wnt and Notch signaling. In support of this, immunohistochemistry and gene expression analysis revealed that both Wnt and Notch signaling pathways were hyperactive in Mtgr1−/− tumors. Furthermore, in human colorectal cancer (CRC) samples MTGR1 was downregulated at both the transcript and protein level. Overall our data indicates that MTGR1 has a context-dependent effect on intestinal tumorigenesis.

MTG16 is a tumor suppressor in colitis-associated carcinoma

MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16-/- mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16-/- mice failed to rescue the Mtg16-/- protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.

717 Loss of MTGR1 Accelerates Intestinal Tumor Burden

Recent genome-wide association studies have uncovered genetic links for a number of complex diseases, but these powerful tools have not yet been widely applied to the clinical management of disease. Here we describe our experience using a genetic approach to uncover newmolecular insights in a young patient presenting with unexplained infections and chronic intestinal inflammation whomwe eventually diagnosed with anti-goblet cell antibody positive autoimmune enterocolopathy. Using a single-nucleotide polymorphism (SNP) array, we identified a deletion of the gene Nuclear Factor of Activated T cells-5 (NFAT5) in one chromosome in the patient and verified this deficiency at the mRNA and protein levels. In mice, NFAT5 has previously been shown to regulate cellular responses to osmotic stress and to play a critical role in influencing differentiation of pro-inflammatory T helper 17 (Th17) lymphocytes. We demonstrate for the first time that human NFAT5 deficiency appears to result in functional defects in both innate and adaptive immunity, with a markedly reduced number of natural killer (NK) cells, and impaired proliferation and cytokine production by T lymphocytes. We confirm the molecular link between immune cell function and NFAT5 deficiency using human cells as well as cells from mice deficient in NFAT5. Together our approach and observations demonstrate the potential power of genetic analyses to facilitate clinical diagnosis and to provide novel mechanistic insights into disease.

Mo1967 Selenoprotein P Suppresses Tumorigenesis Through Both Its Selenium Transport and Redox Activities in a Mouse Model of Colitis-Associated Carcinoma

UC = 0.84 [95%CI 0.61-1.14], p=0.259; OR of CRC + CD = 0.85 [95%CI 0.67-1.06], p= 0.145). There was no difference in age between CRC patients with IBD (UC and CD averaged) and non-IBD patients. In UC patients, we noted increased frequency of stages I-II CRC and decreased frequency of stages III-IV compared to non-IBD cancer patients (p-value=0.038). There were no differences in cancer stage in CD patients (p-value = 0.282). Conclusion: This study provides new information about IBD-CRC in elderly Americans and suggests that UC and CD may not be significant risk factors for CRC. These findings are similar to findings from the Danish population, and may reflect the impact of cancer prevention programs or reveal additional biologic factors related to disease severity and risks. Further analysis of temporal trends in this unique population is ongoing.

112 MTG16 Functions as a Tumor Suppressor in Murine Inflammatory Carcinogenesis

median age was 60 years (range 45-72 and 45-77 for cases and controls respectively). Four cases and 5 controls had primary sclerosing cholangitis (PSC). Case neoplasms included 9 cancers [median size 2.3 (0.8-5) cm; 6/9 (66%) were proximal to splenic flexure; median AJCC stage was I (range I-IIIC)], and 10 dysplastic lesions [8 visible adenomas (dysplasia grade: 3 high, 5 low) with median size 2.3 (1.0-6.2) cm and two flat lesions (1 high grade, 1 low grade) detected by random biopsy]. Univariate and multivariate logistic regression tested the association of marker copy numbers, and potential confounders, to IBD-CRN and cancer. Results: All 3 markers individually showed high discrimination for IBD-CRN: areas under the receiver operating characteristics curves (AUC) with VIM, NDRG4 and EYA4 were 0.91, 0.84 and 0.85, respectively. For cancer the AUC with VIM, NDRG4 and EYA4 were 0.97, 0.94 and 0.95, respectively. Stool assay of VIM alone at 95% specificity yielded a sensitivity for IBD-CRN of 68% (95% CI 43-86%) and for cancer of 89% (95% CI 51-99%). Neither IBD disease duration nor comorbid PSC influencedmarker levels. Combiningmarkers did not improve discrimination. Conclusions: Stool assay of methylated VIM, NDRG4 or EYA4 highly discriminates IBD-CRN cases from IBD controls. These data corroborate our earlier proof-of-concept report of IBD-CRN detection by stool assay of methylated DNA markers. Further studies are indicated to evaluate this noninvasive approach as a complement to endoscopic strategies in IBD surveillance cohorts.

Mo1601 Selenium and the Major Selenium Transporter Selenoprotein P Modify Colitis-Associated Carcinoma

inhibition of Vav1 does not elevate the risk of colitis associated colon cancer. Subsequently, azathioprine induced blockade of Vav1 mediated Rac1 activation in activated T cells exclusively blocks the overwhelming immune reaction in the context of IBD without supporting colorectal tumorigenesis. Innovative immunosuppressive strategies for optimized IBD therapy might therefore concentrate on an even more specific blockade of the Vav1-Rac1 signaling pathway.