Vishruth K. Reddy

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.

BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation

Blood vessel epicardial substance (BVES/Popdc1) is a junctional‐associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial‐to‐mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves–/– mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild‐type (WT) mice. Intercross with Lgr5‐EGFP reporter mice confirmed expansion of the stem cell compartment in Bves–/– mice. To examine stem cell function after BVES deletion, we used ex vivo 3D‐enteroid cultures. Bves–/– enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt‐base columnar “CBC” and “+4” stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves–/– enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves–/– mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves–/– mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage‐responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626–1636

The transcriptional corepressor MTGR1 regulates intestinal secretory lineage allocation

Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene‐related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight‐Twenty‐One family. Given that Mtgr1–/– mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1–/– intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor‐independent 1 (Gfi1) were down‐regulated in Mtgr1–/– whole intestines and Mtgr1–/– enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch‐induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ‐secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1–/– intestine and increased production of goblet and enteroendocrine lineages in Mtgr1–/– mice. GSI increased Paneth cell production in wild‐type mice but failed to do so in Mtgr1–/– mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation.—Parang, B., Rosenblatt, D., Williams, A. D., Washington, M. K., Revetta, F., Short, S. P., Reddy, V. K., Hunt, A., Shroyer, N. F., Engel, M. E., Hiebert, S. W., Williams, C. S., The transcriptional corepressors MTGR1 regulates intestinal secretory lineage allocation. FASEB J. 29, 786–795 (2015). www.fasebj.org

Percutaneous stereotactic radiofrequency lesioning for trigeminal neuralgia: Determination of minimum clinically important difference in pain improvement for patient-reported outcomes

BACKGROUND The Visual Analog Scale (VAS) and the Barrow Neurological Institute Pain Scale (BNI-PS) are 2 patient-reported outcome (PRO) tools frequently used to rate pain from trigeminal neuralgia (TN). Outcomes studies often use these patient-reported outcomes to assess treatment effectiveness, but it is unknown exactly what degree of change in the numerical scores constitutes the minimum clinically important difference (MCID). MCID remains uninvestigated for percutaneous stereotactic radiofrequency lesioning (RFL), a common surgical procedure for TN. OBJECTIVE To determine MCID values for the VAS and BNI-PS in patients undergoing RFL. METHODS Forty-three consecutive patients with TN who underwent RFL by a single surgeon were prospectively assessed with the VAS and BNI-PS preoperatively and 3 years postoperatively. Three anchors were used to assign each patients outcome: satisfaction, willingness to have the surgery again, and Health Transition Index. We then used 3 well-established, anchor-based methods to calculate MCID: average change, minimum detectable change, and change difference. RESULTS Patients experienced substantial improvement in both VAS (9.81 vs 3.35; P < .001) and BNI-PS (4.95 vs 2.44; P < .001) after RFL. The 3 MCID calculation methods generated a range of MCID values for each of the PROs (VAS, 4.13-8.20; BNI-PS, 1.03-3.30). The area under the receiver-operating characteristic curve was greater for BNI-PS compared with VAS for all 3 anchors, indicating that BNI-PS is probably better suited for calculating MCID. CONCLUSION RFL-specific MCID is variable on the basis of the calculation technique. With the use of the minimum detectable change calculation method with the Health Transition Index anchor, the minimum clinically important difference is 4.49 for VAS and 1.16 for BNI-PS after RFL for TN. ABBREVIATIONS AUC, area under the receiver-operating characteristic curveBNI-PS, Barrow Neurological Institute Pain ScaleHTI, Health Transition IndexMCID, minimum clinically important differenceMDC, minimum detectable changePRO, patient-reported outcomeRFL, percutaneous stereotactic radiofrequency lesioningTN, trigeminal neuralgiaVAS, Visual Analog Scale.

BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves−/− mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves−/− mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves−/− mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.

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.

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.