Shenika Poindexter

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.

Transcriptional corepressor MTG16 regulates small intestinal crypt proliferation and crypt regeneration after radiation-induced injury.

Myeloid translocation genes (MTGs) are transcriptional corepressors implicated in development, malignancy, differentiation, and stem cell function. While MTG16 loss renders mice sensitive to chemical colitis, the role of MTG16 in the small intestine is unknown. Histological examination revealed that Mtg16(-/-) mice have increased enterocyte proliferation and goblet cell deficiency. After exposure to radiation, Mtg16(-/-) mice exhibited increased crypt viability and decreased apoptosis compared with wild-type (WT) mice. Flow cytometric and immunofluorescence analysis of intestinal epithelial cells for phospho-histone H2A.X also indicated decreased DNA damage and apoptosis in Mtg16(-/-) intestines. To determine if Mtg16 deletion affected epithelial cells in a cell-autonomous fashion, intestinal crypts were isolated from Mtg16(-/-) mice. Mtg16(-/-) and WT intestinal crypts showed similar enterosphere forming efficiencies when cultured in the presence of EGF, Noggin, and R-spondin. However, when Mtg16(-/-) crypts were cultured in the presence of Wnt3a, they demonstrated higher enterosphere forming efficiencies and delayed progression to mature enteroids. Mtg16(-/-) intestinal crypts isolated from irradiated mice exhibited increased survival compared with WT intestinal crypts. Interestingly, Mtg16 expression was reduced in a stem cell-enriched population at the time of crypt regeneration. This is consistent with MTG16 negatively regulating regeneration in vivo. Taken together, our data demonstrate that MTG16 loss promotes radioresistance and impacts intestinal stem cell function, possibly due to shifting cellular response away from DNA damage-induced apoptosis and towards DNA repair after injury.

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.

51 BVES Orchestrates Pr61α-PP2A Mediated C-Myc Degradation

Background: Blood vessel epicardial substance (BVES) is a tight-junction associated protein that regulates epithelial-to-mesenchymal transition (EMT). BVES is underexpressed in ulcerative colitis (UC), and the BVES promoter is hypermethylated in colitis-associated cancer (CAC). We have recently presented that Bves mice have greater tumor burden and higher grade dysplasia in the AOM/DSS model of CAC. Here we defined the mechanism by which BVES regulates EMT and CAC. Methods: After tumor induction using AOM/DSS, intratumoral RNA was isolated from Bves and WT mice and the transcriptome defined via RNAseq. Tumors were stained by IHC for c-myc and phospho-S62-c-myc and scored using an index based on positive cells per HPF. C-myc protein analysis was conducted in HEK 293T, HCT116, and SW480 cell lines. Enteroid cultures were isolated from 4-week old mice and harvested after 72 hours for RNA and protein. Cells were treated with 100ug/ml of cycloheximide 72 hours after transfection. Polyhis-tagged ubiquitin was co-transfected with HA-c-myc with empty vector or V5-BVES. Ubiquitin was pulled down using Ni-NTA agarose and c-myc was blotted. Transepithelial resistance (TER) was measured daily for 7 days. WST-1 values were recorded daily for 3 days post-plating. Results: RNA-seq analysis using Ingenuity Pathway identified that the c-myc transcriptional network was disrupted in AOM/ DSS Bves tumors. IHC analysis of c-myc and phopsho-S62c-myc showed an increase in Bves tumors (c-myc 2.6± 0.1 vs. 1.8± 0.1 P<0.0001 and phospho-S62-c-myc 2.8± 0.2 vs. 2.1± 0.1 per tumor HPF P<0.01). Bves enteroids had 10-fold upregulation of c-myc protein and a 38-fold increase in the c-myc transcriptional target ornithine decarboxylase. Knocking down BVES in 293T, HCT116, and SW480 cells increased c-myc protein levels by 25%. Cycloheximide treatment after BVES knockdown doubled c-myc half-life. Further, transfection of BVES increased poly-ubiquitylated c-myc suggesting BVES promotes c-myc degradation. Lastly, immunopreciptation demonstrated that BVES and c-myc exist in complex. A yeast-two-hybrid screen identified PR61α, a regulatory subunit of the PP2A family of phosphatases known to promote c-myc degradation, interacts with BVES. We confirmed the BVES:PR61α interaction by directed Y2H and co-IP. PR61α knockdown blocked BVESinduced reduction of c-myc, suggesting BVES requires PR61α to promote c-myc destruction. Caco-2 cell differentiation requires post-translational downregulation of c-myc. When PR61α was knocked down, BVES was unable to increase TER in Caco-2 cells. Further, c-myc promotes proliferation, and PR61α knockdown prevented BVES-induced reduction of WST1. Conclusions: BVES requires PR61α to regulate TER and proliferation, potentially through directing the c-myc downregulation.

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.

323 BVES Contributes to Colonic Wound Healing After Mechanical Injury

G A A b st ra ct s and the affect on intracellular ROS, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine. Results: TNFα-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal levels of autophagy and levels of TNFα-induced autophagy in intestinal epithelial cells. siPHB in epithelial cells induced mitochondrial autophagy via increased intracellular ROS independent of p53 signaling. Inhibition of autophagy during siPHB knockdown exacerbated mitochondrial depolarization and reduced cell viability. Conclusions: Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.