- Email: [email protected]
Mo1950 Epigenetic Regulation of miR-193a-3p in UC-Associated Neoplasia; A Potential Novel Mechanism of Carcinogenesis Involving Il17rd
AGA Abstracts
Results: BEC-40 weeks and BEC-60 weeks had significantly higher proliferation rates compared to BEC-0 weeks and BEC-20 weeks (See Table 1). Although there was no statistically significant difference in proliferation rate between BEC-0 week and 20 week cells, the BEC60 week cells grew 2.75 fold higher compared to BEC-40 week cells. The apoptosis rate showed a reverse pattern with a 50 % suppression in BEC-60 week cells compared to BEC0 week cells. Serum-deprivation induced a 5.4-fold reduction in proliferation rate in BEC60 week cells compared to the BEC-40 week cells. Conclusion: The transformed BEC-60 week cells proliferate at a faster rate than BEC-0 weeks, 20 weeks, and 40 week cells. They also appear to be more resistant to apoptosis as a consequence of B4 exposure. However, proliferation of BEC-60 weeks is most affected by serum deprivation. Increased proliferation, reduced apoptosis, and serum dependence due to chronic B4 exposure in BEC cells are likely properties of dysplasia and neoplasia observed in BE progression to esophageal adenocarcinoma in vivo. We propose that this dynamic novel BEC model may be applied for future chemoprevention studies for BE-carcinogenesis. Table 1: Fold change between different phases of the BEC model in the presence and absence of serum in growth medium
group than in the persistence group. The number of Ki67 positive cell per gland (P = 0.008) and the mean extent of CDX2 (P = 0.027) was lower in the regression group than in the persistence group. Conclusions: The regression of BE was frequent than expected in Korea. The immunohistochemical detection of mucin phenotype, grade of intestinal metaplasia, Ki67 and CDX2 expression in Barrett's mucosa could be useful as a predictable marker for regression of IM of BE. Table 1. Comparison of immunohistochemical results between regression and persistence group
SIM, specialized intestinal metaplasia; *, n =11 in MUC and CDX2 immunohistochemistry Mo1949 Mo1947 VEGF/VEGFR-2 Signaling Promotes Tumorigenesis in Colitis-Associated Carcinoma Through Inactivation of Premature Senescence in Intestinal Epithelial Cells Sebastian Foersch, Tobias Sperka, Karl L. Rudolph, Georg Breier, Stefan J. Wirtz, Markus F. Neurath, Maximilian J. Waldner
Co-Treatment With Statin May Prevent Development of Barrett's Esophagus Among Japanese Patients Taking Low Doses of Aspirin Tomoko Kanzaki, Akiko Shiotani, Tomoari Kamada, Hiroshi Imamura, Noriaki Manabe, Hiroaki Kusunoki, Tomonari Kimura, Yoshinori Fujimura, Takashi Sakakibara, Jiro Hata, Ken Haruma
Introduction: Colitis-associated cancer (CAC) is a dreaded complication in patients with inflammatory bowel diseases (IBD) and poses a great clinical challenge. Recent data highlight the importance of VEGF/VEGFR-2 signaling not only in angiogenesis, but also as an autocrine proliferation factor for the tumor cells themselves. We examined the molecular mechanisms of this pathway in different models of CAC and colorectal cancer (CRC). Materials and Methods: AOM/DSS was performed in wild-type mice and mice with a conditional knockout of VEGFR-2 in intestinal epithelial cells (VEGFR-2 ΔIEC). Endoscopic surveillance was used to score the degree of inflammation and tumor mass. Cryosections, paraffin-embedded sections, RNA-isolation, protein-isolation were done for (immuno)histopathologic evaluation, western blot, real-time-PCR and co-immunoprecipitation. Different colorectal cancer cell lines were used for in vitro evaluation of cell growth and senescence (SA-beta-Gal staining) after treatment with PI3K and VEGFR-2 inhibitors. Results: VEGFR ΔIEC mice showed significantly lower tumor-number and -mass compared to wild-type mice, while inflammation was not affected. VEGFR-2ΔIEC tumors showed a distinctly different histomorphology and were better differentiated. While the number of apoptotic cells was not significantly different in both groups, markers for premature senescence (Ki67 negativity, p21, pH2A.X, Cyclin of G1-Phase) were upregulated in VEGFR-2 ΔIEC tumors. In human colorectal cell lines, VEGFR2-dependent activation of the PI3K / Akt pathway could be identified using western blot and co-immunoprecipitation causing a phosphorylation-dependent inhibition of p21. Inhibition of the PI3K / Akt pathway led to senescence induction (SA-beta-Gal expression) in vitro. Conclusion: Our results highlight the role of VEGF/VEGFR-2 signaling as an independent tumor growth factor completely irrespective of angiogenesis. Premature senescence as an anti-tumor property, which can be induced by chronic inflammation in CED, is inactivated over the VEGFR-2 / PI3K / Akt pathway and blockade of p21. This might be a central mechanism by which chronic inflammation and tumor development are connected and could pose a further rationale for using angiogenesis- and PI3K inhibitors in the therapy of colitis-associated cancer.
Background: The recent reports suggest a possible risk for erosive esophagitis with aspirin use. Moreover, a recent clinical study (Gastroenterology. 2010; 138: 2260) and in vivo previous studies suggested a possible reduced risk of esophageal adenocarcinoma with proton pump inhibitors (PPI), non-steroidal anti-inflammatory drugs (NSAIDs)/aspirin and statins. A limited number of clinical studies investigated the effect of aspirin and statin use on the development of Barrett's esophagus. Aim: To investigate the association between Barrett's esophagus with combined medicines including statin and other clinical parameters among patients taking low doses of aspirin. Methods: Patients taking 100 mg enteric-coated aspirin for cardiovascular diseases who underwent upper GI endoscopy with clear endoscopic images of esophagogastric (EG) junction were enrolled. The endoscopic records were evaluated using Prague C&M Criteria for Barrett's esophagus, and Barrett's esophagus as positive when the segment length of metaplastic columnar of the lower esophagus was circumferentially 1 cm and longer. The severity of erosive esophagitis was graded from A-D according to the Los Angeles (LA) classification. H. pylori IgG antibody was measured by ELISA. Results: 411 patients (281 men and 130 women, mean age 71.4±8.6 years old) were enrolled. The prevalence of erosive esophagitis and Barrett's esophagus was 7.1% and 17.8%, respectively. The frequency of the patients with both was 1.9 %. The frequency of the patients taking statin in the Barrett's esophagus group was significantly lower (34.2% vs. 49.9 %, p=0.02) than that in the patients without Barrett's esophagus. Barrett's esophagus was significantly associated with hiatus hernia (adjusted OR 2.79, 95% C.I. 1.50-5.20), active smoking (adjusted OR 2.50, 95% C.I. 1.25-4.96), and co-treatment with statin (adjusted OR 0.51, 95% C.I. 0.30-0.88), but not any other medicine. Conclusion: Co-treatment with statin may prevent and active smoking may cause development of Barrett's esophagus among Japanese patients taking low doses of aspirin. Mo1948
Mo1950
Frequent Regression Rate and Predictable Marker of Regression of Barrett's Esophagus in Korean Hyun Jin Jo, Nayoung Kim, Hye Seung Lee, Ryoung Hee Nam, Hyun Chang, Dong Ho Lee, Hyun Chae Jung
Epigenetic Regulation of miR-193a-3p in UC-Associated Neoplasia; A Potential Novel Mechanism of Carcinogenesis Involving Il17rd Joel Pekow, Katherine Meckel, Urszula Dougherty, Reba Mustafi, Marc Bissonnette
Background/Aims: Previously it has been reported that the normalization rate of specialized intestinal metaplasia (SIM) reached up to 30% in short segment Barrett's esophagus (BE) in Western countries, where the prevalence of BE is rather high. In Korea, the prevalence of BE is very low, such as below 1%, and most of BE was known as the short segment BE, but there has been no report about regression of BE. The aim of this study was to determine the regression rate of BE and predictable markers related to regression, including clinical/ demographic and histopathologic factors. Methods: Total 35 patients diagnosed as BE at initial biopsy were consecutively enrolled and the 25 patients underwent endoscopic surveillance and biopsy. The mean follow-up period was 33.4 months and the mean number of surveillance endoscopy was 2.4 times. If the SIM was lost at any point of surveillance and did not recur, the cases were grouped as the regression group. While, if the SIM was not disappeared, then the cases were categorized as persistence group. The proportion of SIM in total columnar cells was graded as grade 0 (0%), grade 1 (1-29%), grade 2 (30-69%), grade 3 (.70%). The gastric or intestinal mucin phenotype was decided using immunohistochemical analysis for MUC2, MUC5AC and MUC6. To assess the cell proliferation indexes and the degree of intestinal maturation, immunohistochemistry for Ki67 and CDX2 were performed. Results: 11 (44 %) patients get the regression of BE. The clinical and demographic factors showed no difference between the regression and persistence group. The lower grade of SIM (P ,0.001) and gastric mucin phenotype (P = 0.018) were frequent in the regression
AGA Abstracts
Background: miR-193a-3p functions as a tumor suppressor in some cancers, although downregulation was not reported in sporadic or colitis-associated colon cancer. In prior microarray experiments, we identified significant down regulations of miR-193a-3p in ulcerative colitis (UC)-associated neoplasia lesions compared to both UC without dysplasia and normal controls. To explore the significance of these findings, we investigated a larger cohort of patients and explored regulatory mechanisms as well as miRNA targets in cell culture. Methods: We extracted total RNA and DNA from formalin fixed paraffin embedded colonic tissues from 12 controls (N), 10 UC patients without dysplasia (UC), 10 UC dysplastic lesions (UCD: 4 HGD and 6 CRC), and 12 adjacent non-dysplastic tissues in UC patients harboring remote neoplasia (UCrD). miR-193a-3p was evaluated by qPCR, and miR-193a methylation was investigated in bisulfite-treated DNA using methylation-specific high resolution melting analysis. Melting curves were normalized using Roche Gene Scanning Software and a differential profile was evaluated by comparing change in fluorescence to methylated standards. LoVo, HCT116, and DLD-1 cells were treated with 5-aza-2'deoxycytidine (5AZA) daily for 5 days and expression of pri-miR-193a and mature miR-193a-3p was measured. HCT116 cells were transfected with 193a-3p or scrambled-control and expression levels of in silico predicted targets, IL17RD and KRAS, were assessed by Westerns. Results: miR-193a-3p expression was down-regulated 2.9-fold in UC (p=0.04), 3.3-fold in UCrD
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MMP9 overexpressing cells compared to vector. This was supported by WB indicating that MMP9 overexpressing cells compared to vector have significantly lower levels of MDC1 (60±7.6 percent). Interestingly, MMP9 overexpressing cells showed significantly decreased levels of MLH1 (55±8.2 percent). Conclusion: MMP9 mediates decreased proliferation and cell cycle arrest via activation of p21WAF1/Cip1. Also a significant decrease in DNA damage and mismatch repair genes contributes to MMP9 mediated protection in CAC. These outcomes will fundamentally advance the designing of therapeutic targets in the field of CAC. Mo1953 Genetic Diversity of a Single H. pylori Strain Over a Five-Year Period in a Primate Model: Effect of Long Term Colonization and of a Dietary Carcinogen Hui Liu, Beth M. Carpenter, Cristina Semino-Mora, Nina Salama, Thomas Borén, Andre Dubois, Douglas S. Merrell Background. H. pylori isolates from different individuals exhibit substantial genetic diversity and have been shown to evolve during the course of infection. However, the kinetics of bacterial evolution have not been studied in vivo over an extended period of time. Aims. The present study investigated the effect of long-term H. pylori infection with or without oral administration of the nitrosating carcinogen ethyl-nitro-nitrosoguanidine (ENNG) on H. pylori strain USU101 in Rhesus macaques. Methods. 12 macaques were inoculated with H. pylori USU101 alone (H) or in combination with ENNG administration (EH), and gastroscopies were performed at regular intervals over the course of 5 years. Biopsies were cultured for H. pylori at regular intervals, and single colony isolates were analyzed by DNA fingerprinting. Microarray based comparative genomic hybridizations (aCGH) were performed to identify missing genes and the results were confirmed by PCR. Temporal samples spanning 5 years were analyzed to observe when and how the gene for the outer membrane protein BabA changed. To evaluate BabA expression, changes in Leb-binding were evaluated by radioimmunoassay and total BabA protein was quantitated using Western blot analysis. Shotgun sequencing of some output strains was conducted. Results. At 1-year, all output isolates were identical to the input strain as assessed by DNA fingerprinting. At 5-year, different H. pylori fingerprints were observed in both groups and, as previously reported, intramucosal neoplasia was observed only in EH macaques. aCGH revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes that included alteration of the outer membrane protein (OMP) repertoire in EH and H isolates. At the DNA level, these changes started at 18 month post inoculation, but changes in protein level appeared much earlier: Western blots and colony screening for Leb-binding showed that the levels of BabA protein were dramatically reduced within weeks of H. pylori infection. Currently, we are investigating potential changes in the promoter of babA, which could be responsible for the decreased expression over time. Conclusion. Long term colonization of the gastric niche can lead to specific H. pylori genetic changes that appear to reflect responses to the host mucosal properties, but not to administration of a dietary carcinogen. Ongoing studies continue to determine the mechanism of in vivo bacterial evolution.
Mo1951 Sensitive Quantitative CpG Methylation Testing in Random Colonic Biopsies for Risk Assessment of Coexisting Dysplasia in Ulcerative Colitis Patients Antonia R. Sepulveda, Takeshi Uehara, Dara L. Aisner, Yuan Yao, Miguel Regueiro, Wen Yan, Junya Masumoto, John W. Tobias, Khushboo Jhala, Roger Day, Franz Fogt, Hiroyoshi Ota, Jorge Sepulveda Background: Dysplasia and cancer in ulcerative colitis (UC) occur in a field defect that characterizes the background colitis which may carry epigenetic changes in cancer related genes leading to altered gene regulation and neoplasia. Aims: To identify a panel of CpG methylation loci to help assess for the presence of dysplasia by testing DNA from random colonic mucosa tissue. CpG loci that undergo higher levels of methylation in the background non-dysplastic colitic mucosa of UC patients with co-existing dysplasia as compared to colitis of patients without dysplasia were selected for the panel. Design: CpG methylated loci were selected by comparing methylation levels in UC-dysplasia and mucosa with colitis with GoldenGate methylation cancer panel bead arrays, which interrogate CpG sites from 807 cancer-related genes. Additional CpG methylation targets were selected among genes known to undergo CpG methylation in UC-associated neoplasia (MLH1, MGMT, p16 and TGFBR3). CpG methylation levels in promoter regions of selected target genes were determined in the mucosa of 49 colectomy specimens (16 colons with normal mucosa from patients without UC, 12 from UC patients without dysplasia or cancer, and 21 from UC patients with dysplasia). CpG methylation levels were determined in DNA extracted from paraffin sections and bisulfate-modified DNA was used for quantitative SYBR green methylation specific PCR (QMSP). Methylation levels 1% or greater of reference were considered positive. Results: GoldenGate array data showed that methylation of GAS7, SFRP1 and DCC was significantly higher in dysplasia as compared to UC background non-neoplastic mucosa with colitis. Methylation levels of all 7 genes (GAS7, SFRP1, DCC, MLH1, MGMT, p16 and TGFBR3) were determined in the 49 colectomy specimens. ROC analysis was performed to compare the diagnostic ability of methylation levels at the 7 loci in background mucosa without dysplasia to identify concurrent dysplasia in UC patients. The marker with the highest area under the ROC (AU) was GAS7 (AU=0.89), followed by MLH1 (AU=0.79). When the different combinations of markers were tested in a logistic regression model, the model using just 5 loci (GAS7, MLH1, p16, SFRP1, and MGMT) had the highest diagnostic performance (AU = 0.95). With this 5-loci model, specificity was 100% and sensitivity was 90% for the presence of concurrent dysplasia. Conclusions: Novel CpG methylation markers were identified in UC-dysplasia by GoldenGate arrays. Our data suggest that a numerical score derived from the combination of quantitative CpG methylation levels of a small number of CpG gene target loci in random mucosal biopsies may be highly specific and sensitive for the prediction of dysplasia in UC patients. This test may be useful to refine the selection criteria for colonoscopy surveillance and treatment approach in UC patients.
Mo1954 DNA Mismatch Repair Deficiency in Inflammatory Bowel Disease Associated Colorectal Carcinomas Jeremy S. Ditelberg, Shelley Karl, Mark Redston Background: Colorectal cancer (CRC) occurring in the setting of Inflammatory Bowel Disease (IBD) is relatively uncommon. Compared to sporadic CRC, the molecular pathogenesis of IBD-associated CRC has some distinctive features, particularly the timing of acquisition of genetic changes during progression through high grade dysplasia. DNA mismatch repair (MMR) deficient sporadic CRC is a well-defined entity comprised of Lynch syndromeassociated cancers and cancers characterized by a CpG island methylator phenotype (CIMP). The latter cancers characteristically arise in sessile serrated adenomas/polyps. The role of MMR deficiency in IBD CRC is less well established. We undertook this study to determine the frequency and clinical pathologic associations of MMR deficiency in IBD-associated neoplasia. Methods: Patients with CRC were selected from the pathology database of Miraca Life Sciences. The database includes demographic and clinical information, summary of the endoscopic report, biopsy location, and the histopathologic report for each biopsy specimen. Cases of CRC with a report date from 11/1/2007 to 11/30/2011 were extracted. From these cases, patients with a history of Ulcerative Colitis (UC) and Crohn Disease (CD) were extracted. Immunohistochemical (IHC) staining for MMR proteins MLH1, MSH2, MSH6 and PMS2 were performed on the CRC and interpreted as intact (positive staining) or deficient (loss of staining). Results: Biopsies from 917,599 colonoscopies were interpreted on 878,835 patients (51% female, 49% male) during the study period. Of these, 16,631 unique patients (48% female, 52% male) had histological and clinical features of UC while 5,736 patients had histological and clinical features of CD. During this period, 6,211 CRCs were diagnosed (52% male, 48% female). Of these CRCs, 22 occurred in patients with UC (prevalence of 22/16,631 = 0.13%), and 5 occurred in patients with CD (prevalence of 5/ 5,736 = 0.09%). 19 IBD patients were male (70.4%) and 8 were female (29.6%). The mean age was 58 years (median 56 years, standard deviation 13.5 years, range 33 to 84 years). Of these tumors, 14 (51.9%) were moderately differentiated (MD) and 13 (48.1%) were poorly differentiated (PD). IHC staining for MMR proteins showed MLH1 and PMS2 deficiency abnormalities in 3/27 (11%) patients, all of whom had UC. These 3 patients were 65, 77 and 79 years old and had CRC in the cecum, rectum and left colon, respectively. MSH2 and MSH6 expression were intact in all cases. Conclusions: MMR protein deficiency in the setting of IBD associated CRC appears to be less frequent than in the sporadic setting. Demographic characteristics suggest that it is likely due to MLH1 promoter methylation. Additional studies are needed to further define the mechanism. CRC occurring in the setting of IBD is rare, occurs predominantly in males and tends to be PD.
Mo1952 Matrix Metalloprotease (MMP9) Mediates Protection in Colitis Associated Cancer (CAC) by Modulating Cell Cycle Arrest via p21WAF1 Lewins Walter, Vaishali Pannu, Ritu Aneja, Pallavi Garg Background and Aim: Individuals with IBD have increased risk of developing colitis associated cancer (CAC). However, the pathogenesis of CAC is incompletely understood. Matrix metalloproteinases (MMPs) are zinc-dependent neutral endopeptidases that participate in degradation of extra cellular matrix proteins that occurs in both normal tissue remodeling and a variety of pathological processes including inflammation and cancer. MMP9 protein expression and activity is undetectable in normal tissues but is highly expressed in inflammatory state. We have shown that despite being a mediator of inflammation epithelial cell derived MMP9 plays a protective role in the development of CAC. Aim of this study is to comprehend the mechanism by which MMP9 mediates protection in CAC. Methods: MMP9-/- and wild type mice (WT) with and without CAC were used as in vivo model. Stably transfected HCT116 cells with pEGFP plasmid with & without MMP9 and mouse embryonic fibroblast cells (MEFs) obtained from MMP9-/- and WTs were used as in vitro models. Cell cycle events were analyzed by flow cytometry. Immunostaining was performed to detect DNA damage. Results: MMP9-/- mice exhibited significantly decreased expression of p53 and p21WAF1/ Cip1 compared to WTs in CAC. HCT116 cells overexpressing MMP9 showed a significant decrease in proliferation at 48 and 72 hours compared to vector. There was significant increase in p53 (8±0.32 fold) and p21WAFI/Cip1 (15±0.86 fold) expressions but a significant decrease in cyclinD1 expression (45±2.34 percent) in MMP9 overexpressing cells compared to vector. Overexpressing MMP9 cells exhibited a significant decrease in cyclinA expression (30±8.5 percent) but there was no significant change in cyclin E expression compared to vector. Results obtained from MEFs supported these findings, as irradiated MMP9-/- MEFs have significant decrease in p53 and p21WAF1/Cip1 expressions compared to WT MEFs. MMP9-/- MEFs also indicated a significant increase in cyclinA (3±0.5 fold) protein levels with subtle difference in cyclinE protein levels. Flow cytometry of MMP9 overexpressing cells showed a significant arrest of 8.5% in S phase of cell cycle compared to vector. DNA double strand breaks detected by immunostaining of H2AX showed less DNA damage in
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AGA Abstracts
AGA Abstracts
(p=0.03), and 5.3-fold in UCD (p=0.003) compared to normal controls. Compared to UC without dysplasia, miR-193a-3p was down-regulated 1.2-fold in UCrD (p=0.7) and 1.9-fold in UCD (p=0.05). UCD samples demonstrated increased methylation compared to normal controls and UC without neoplasia. There was a linear trend with decreased expression and increased methylation across all samples. 5-AZA treatment resulted in increased pri-miR193a transcript abundance in DLD1 (2.1-fold, p= 0.002), HCT116 cells (2.2-fold, p=0.02), and LoVo cells (1.2-fold, p=0.7), without significant change in mature miR-193a-3p expression. Transfection of 20nM and 50nM of miR-193a-3p in HCT116 cells down-regulated IL17RD, but not KRAS. Discussion: The gene encoding miR-193a-3p is methylated and its expression is down-regulated in IBD-associated neoplasia, suggesting that miR-193a-3p is regulated epigenetically. We identified IL17RD, an orphan receptor that potentiates IL-17 signaling, as a novel target of miR-193a-3p. As IL-17 signaling is up-regulated in inflammation and carcinogenesis, we postulate that down-regulation of miR-193a-3p contributes to cancer development in IBD via increased IL17RD.
Results: BEC-40 weeks and BEC-60 weeks had significantly higher proliferation rates compared to BEC-0 weeks and BEC-20 weeks (See Table 1). Although there was no statistically significant difference in proliferation rate between BEC-0 week and 20 week cells, the BEC60 week cells grew 2.75 fold higher compared to BEC-40 week cells. The apoptosis rate showed a reverse pattern with a 50 % suppression in BEC-60 week cells compared to BEC0 week cells. Serum-deprivation induced a 5.4-fold reduction in proliferation rate in BEC60 week cells compared to the BEC-40 week cells. Conclusion: The transformed BEC-60 week cells proliferate at a faster rate than BEC-0 weeks, 20 weeks, and 40 week cells. They also appear to be more resistant to apoptosis as a consequence of B4 exposure. However, proliferation of BEC-60 weeks is most affected by serum deprivation. Increased proliferation, reduced apoptosis, and serum dependence due to chronic B4 exposure in BEC cells are likely properties of dysplasia and neoplasia observed in BE progression to esophageal adenocarcinoma in vivo. We propose that this dynamic novel BEC model may be applied for future chemoprevention studies for BE-carcinogenesis. Table 1: Fold change between different phases of the BEC model in the presence and absence of serum in growth medium
group than in the persistence group. The number of Ki67 positive cell per gland (P = 0.008) and the mean extent of CDX2 (P = 0.027) was lower in the regression group than in the persistence group. Conclusions: The regression of BE was frequent than expected in Korea. The immunohistochemical detection of mucin phenotype, grade of intestinal metaplasia, Ki67 and CDX2 expression in Barrett's mucosa could be useful as a predictable marker for regression of IM of BE. Table 1. Comparison of immunohistochemical results between regression and persistence group
SIM, specialized intestinal metaplasia; *, n =11 in MUC and CDX2 immunohistochemistry Mo1949 Mo1947 VEGF/VEGFR-2 Signaling Promotes Tumorigenesis in Colitis-Associated Carcinoma Through Inactivation of Premature Senescence in Intestinal Epithelial Cells Sebastian Foersch, Tobias Sperka, Karl L. Rudolph, Georg Breier, Stefan J. Wirtz, Markus F. Neurath, Maximilian J. Waldner
Co-Treatment With Statin May Prevent Development of Barrett's Esophagus Among Japanese Patients Taking Low Doses of Aspirin Tomoko Kanzaki, Akiko Shiotani, Tomoari Kamada, Hiroshi Imamura, Noriaki Manabe, Hiroaki Kusunoki, Tomonari Kimura, Yoshinori Fujimura, Takashi Sakakibara, Jiro Hata, Ken Haruma
Introduction: Colitis-associated cancer (CAC) is a dreaded complication in patients with inflammatory bowel diseases (IBD) and poses a great clinical challenge. Recent data highlight the importance of VEGF/VEGFR-2 signaling not only in angiogenesis, but also as an autocrine proliferation factor for the tumor cells themselves. We examined the molecular mechanisms of this pathway in different models of CAC and colorectal cancer (CRC). Materials and Methods: AOM/DSS was performed in wild-type mice and mice with a conditional knockout of VEGFR-2 in intestinal epithelial cells (VEGFR-2 ΔIEC). Endoscopic surveillance was used to score the degree of inflammation and tumor mass. Cryosections, paraffin-embedded sections, RNA-isolation, protein-isolation were done for (immuno)histopathologic evaluation, western blot, real-time-PCR and co-immunoprecipitation. Different colorectal cancer cell lines were used for in vitro evaluation of cell growth and senescence (SA-beta-Gal staining) after treatment with PI3K and VEGFR-2 inhibitors. Results: VEGFR ΔIEC mice showed significantly lower tumor-number and -mass compared to wild-type mice, while inflammation was not affected. VEGFR-2ΔIEC tumors showed a distinctly different histomorphology and were better differentiated. While the number of apoptotic cells was not significantly different in both groups, markers for premature senescence (Ki67 negativity, p21, pH2A.X, Cyclin of G1-Phase) were upregulated in VEGFR-2 ΔIEC tumors. In human colorectal cell lines, VEGFR2-dependent activation of the PI3K / Akt pathway could be identified using western blot and co-immunoprecipitation causing a phosphorylation-dependent inhibition of p21. Inhibition of the PI3K / Akt pathway led to senescence induction (SA-beta-Gal expression) in vitro. Conclusion: Our results highlight the role of VEGF/VEGFR-2 signaling as an independent tumor growth factor completely irrespective of angiogenesis. Premature senescence as an anti-tumor property, which can be induced by chronic inflammation in CED, is inactivated over the VEGFR-2 / PI3K / Akt pathway and blockade of p21. This might be a central mechanism by which chronic inflammation and tumor development are connected and could pose a further rationale for using angiogenesis- and PI3K inhibitors in the therapy of colitis-associated cancer.
Background: The recent reports suggest a possible risk for erosive esophagitis with aspirin use. Moreover, a recent clinical study (Gastroenterology. 2010; 138: 2260) and in vivo previous studies suggested a possible reduced risk of esophageal adenocarcinoma with proton pump inhibitors (PPI), non-steroidal anti-inflammatory drugs (NSAIDs)/aspirin and statins. A limited number of clinical studies investigated the effect of aspirin and statin use on the development of Barrett's esophagus. Aim: To investigate the association between Barrett's esophagus with combined medicines including statin and other clinical parameters among patients taking low doses of aspirin. Methods: Patients taking 100 mg enteric-coated aspirin for cardiovascular diseases who underwent upper GI endoscopy with clear endoscopic images of esophagogastric (EG) junction were enrolled. The endoscopic records were evaluated using Prague C&M Criteria for Barrett's esophagus, and Barrett's esophagus as positive when the segment length of metaplastic columnar of the lower esophagus was circumferentially 1 cm and longer. The severity of erosive esophagitis was graded from A-D according to the Los Angeles (LA) classification. H. pylori IgG antibody was measured by ELISA. Results: 411 patients (281 men and 130 women, mean age 71.4±8.6 years old) were enrolled. The prevalence of erosive esophagitis and Barrett's esophagus was 7.1% and 17.8%, respectively. The frequency of the patients with both was 1.9 %. The frequency of the patients taking statin in the Barrett's esophagus group was significantly lower (34.2% vs. 49.9 %, p=0.02) than that in the patients without Barrett's esophagus. Barrett's esophagus was significantly associated with hiatus hernia (adjusted OR 2.79, 95% C.I. 1.50-5.20), active smoking (adjusted OR 2.50, 95% C.I. 1.25-4.96), and co-treatment with statin (adjusted OR 0.51, 95% C.I. 0.30-0.88), but not any other medicine. Conclusion: Co-treatment with statin may prevent and active smoking may cause development of Barrett's esophagus among Japanese patients taking low doses of aspirin. Mo1948
Mo1950
Frequent Regression Rate and Predictable Marker of Regression of Barrett's Esophagus in Korean Hyun Jin Jo, Nayoung Kim, Hye Seung Lee, Ryoung Hee Nam, Hyun Chang, Dong Ho Lee, Hyun Chae Jung
Epigenetic Regulation of miR-193a-3p in UC-Associated Neoplasia; A Potential Novel Mechanism of Carcinogenesis Involving Il17rd Joel Pekow, Katherine Meckel, Urszula Dougherty, Reba Mustafi, Marc Bissonnette
Background/Aims: Previously it has been reported that the normalization rate of specialized intestinal metaplasia (SIM) reached up to 30% in short segment Barrett's esophagus (BE) in Western countries, where the prevalence of BE is rather high. In Korea, the prevalence of BE is very low, such as below 1%, and most of BE was known as the short segment BE, but there has been no report about regression of BE. The aim of this study was to determine the regression rate of BE and predictable markers related to regression, including clinical/ demographic and histopathologic factors. Methods: Total 35 patients diagnosed as BE at initial biopsy were consecutively enrolled and the 25 patients underwent endoscopic surveillance and biopsy. The mean follow-up period was 33.4 months and the mean number of surveillance endoscopy was 2.4 times. If the SIM was lost at any point of surveillance and did not recur, the cases were grouped as the regression group. While, if the SIM was not disappeared, then the cases were categorized as persistence group. The proportion of SIM in total columnar cells was graded as grade 0 (0%), grade 1 (1-29%), grade 2 (30-69%), grade 3 (.70%). The gastric or intestinal mucin phenotype was decided using immunohistochemical analysis for MUC2, MUC5AC and MUC6. To assess the cell proliferation indexes and the degree of intestinal maturation, immunohistochemistry for Ki67 and CDX2 were performed. Results: 11 (44 %) patients get the regression of BE. The clinical and demographic factors showed no difference between the regression and persistence group. The lower grade of SIM (P ,0.001) and gastric mucin phenotype (P = 0.018) were frequent in the regression
AGA Abstracts
Background: miR-193a-3p functions as a tumor suppressor in some cancers, although downregulation was not reported in sporadic or colitis-associated colon cancer. In prior microarray experiments, we identified significant down regulations of miR-193a-3p in ulcerative colitis (UC)-associated neoplasia lesions compared to both UC without dysplasia and normal controls. To explore the significance of these findings, we investigated a larger cohort of patients and explored regulatory mechanisms as well as miRNA targets in cell culture. Methods: We extracted total RNA and DNA from formalin fixed paraffin embedded colonic tissues from 12 controls (N), 10 UC patients without dysplasia (UC), 10 UC dysplastic lesions (UCD: 4 HGD and 6 CRC), and 12 adjacent non-dysplastic tissues in UC patients harboring remote neoplasia (UCrD). miR-193a-3p was evaluated by qPCR, and miR-193a methylation was investigated in bisulfite-treated DNA using methylation-specific high resolution melting analysis. Melting curves were normalized using Roche Gene Scanning Software and a differential profile was evaluated by comparing change in fluorescence to methylated standards. LoVo, HCT116, and DLD-1 cells were treated with 5-aza-2'deoxycytidine (5AZA) daily for 5 days and expression of pri-miR-193a and mature miR-193a-3p was measured. HCT116 cells were transfected with 193a-3p or scrambled-control and expression levels of in silico predicted targets, IL17RD and KRAS, were assessed by Westerns. Results: miR-193a-3p expression was down-regulated 2.9-fold in UC (p=0.04), 3.3-fold in UCrD
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MMP9 overexpressing cells compared to vector. This was supported by WB indicating that MMP9 overexpressing cells compared to vector have significantly lower levels of MDC1 (60±7.6 percent). Interestingly, MMP9 overexpressing cells showed significantly decreased levels of MLH1 (55±8.2 percent). Conclusion: MMP9 mediates decreased proliferation and cell cycle arrest via activation of p21WAF1/Cip1. Also a significant decrease in DNA damage and mismatch repair genes contributes to MMP9 mediated protection in CAC. These outcomes will fundamentally advance the designing of therapeutic targets in the field of CAC. Mo1953 Genetic Diversity of a Single H. pylori Strain Over a Five-Year Period in a Primate Model: Effect of Long Term Colonization and of a Dietary Carcinogen Hui Liu, Beth M. Carpenter, Cristina Semino-Mora, Nina Salama, Thomas Borén, Andre Dubois, Douglas S. Merrell Background. H. pylori isolates from different individuals exhibit substantial genetic diversity and have been shown to evolve during the course of infection. However, the kinetics of bacterial evolution have not been studied in vivo over an extended period of time. Aims. The present study investigated the effect of long-term H. pylori infection with or without oral administration of the nitrosating carcinogen ethyl-nitro-nitrosoguanidine (ENNG) on H. pylori strain USU101 in Rhesus macaques. Methods. 12 macaques were inoculated with H. pylori USU101 alone (H) or in combination with ENNG administration (EH), and gastroscopies were performed at regular intervals over the course of 5 years. Biopsies were cultured for H. pylori at regular intervals, and single colony isolates were analyzed by DNA fingerprinting. Microarray based comparative genomic hybridizations (aCGH) were performed to identify missing genes and the results were confirmed by PCR. Temporal samples spanning 5 years were analyzed to observe when and how the gene for the outer membrane protein BabA changed. To evaluate BabA expression, changes in Leb-binding were evaluated by radioimmunoassay and total BabA protein was quantitated using Western blot analysis. Shotgun sequencing of some output strains was conducted. Results. At 1-year, all output isolates were identical to the input strain as assessed by DNA fingerprinting. At 5-year, different H. pylori fingerprints were observed in both groups and, as previously reported, intramucosal neoplasia was observed only in EH macaques. aCGH revealed that long term persistence of USU101 in the macaque stomach was associated with specific whole gene changes that included alteration of the outer membrane protein (OMP) repertoire in EH and H isolates. At the DNA level, these changes started at 18 month post inoculation, but changes in protein level appeared much earlier: Western blots and colony screening for Leb-binding showed that the levels of BabA protein were dramatically reduced within weeks of H. pylori infection. Currently, we are investigating potential changes in the promoter of babA, which could be responsible for the decreased expression over time. Conclusion. Long term colonization of the gastric niche can lead to specific H. pylori genetic changes that appear to reflect responses to the host mucosal properties, but not to administration of a dietary carcinogen. Ongoing studies continue to determine the mechanism of in vivo bacterial evolution.
Mo1951 Sensitive Quantitative CpG Methylation Testing in Random Colonic Biopsies for Risk Assessment of Coexisting Dysplasia in Ulcerative Colitis Patients Antonia R. Sepulveda, Takeshi Uehara, Dara L. Aisner, Yuan Yao, Miguel Regueiro, Wen Yan, Junya Masumoto, John W. Tobias, Khushboo Jhala, Roger Day, Franz Fogt, Hiroyoshi Ota, Jorge Sepulveda Background: Dysplasia and cancer in ulcerative colitis (UC) occur in a field defect that characterizes the background colitis which may carry epigenetic changes in cancer related genes leading to altered gene regulation and neoplasia. Aims: To identify a panel of CpG methylation loci to help assess for the presence of dysplasia by testing DNA from random colonic mucosa tissue. CpG loci that undergo higher levels of methylation in the background non-dysplastic colitic mucosa of UC patients with co-existing dysplasia as compared to colitis of patients without dysplasia were selected for the panel. Design: CpG methylated loci were selected by comparing methylation levels in UC-dysplasia and mucosa with colitis with GoldenGate methylation cancer panel bead arrays, which interrogate CpG sites from 807 cancer-related genes. Additional CpG methylation targets were selected among genes known to undergo CpG methylation in UC-associated neoplasia (MLH1, MGMT, p16 and TGFBR3). CpG methylation levels in promoter regions of selected target genes were determined in the mucosa of 49 colectomy specimens (16 colons with normal mucosa from patients without UC, 12 from UC patients without dysplasia or cancer, and 21 from UC patients with dysplasia). CpG methylation levels were determined in DNA extracted from paraffin sections and bisulfate-modified DNA was used for quantitative SYBR green methylation specific PCR (QMSP). Methylation levels 1% or greater of reference were considered positive. Results: GoldenGate array data showed that methylation of GAS7, SFRP1 and DCC was significantly higher in dysplasia as compared to UC background non-neoplastic mucosa with colitis. Methylation levels of all 7 genes (GAS7, SFRP1, DCC, MLH1, MGMT, p16 and TGFBR3) were determined in the 49 colectomy specimens. ROC analysis was performed to compare the diagnostic ability of methylation levels at the 7 loci in background mucosa without dysplasia to identify concurrent dysplasia in UC patients. The marker with the highest area under the ROC (AU) was GAS7 (AU=0.89), followed by MLH1 (AU=0.79). When the different combinations of markers were tested in a logistic regression model, the model using just 5 loci (GAS7, MLH1, p16, SFRP1, and MGMT) had the highest diagnostic performance (AU = 0.95). With this 5-loci model, specificity was 100% and sensitivity was 90% for the presence of concurrent dysplasia. Conclusions: Novel CpG methylation markers were identified in UC-dysplasia by GoldenGate arrays. Our data suggest that a numerical score derived from the combination of quantitative CpG methylation levels of a small number of CpG gene target loci in random mucosal biopsies may be highly specific and sensitive for the prediction of dysplasia in UC patients. This test may be useful to refine the selection criteria for colonoscopy surveillance and treatment approach in UC patients.
Mo1954 DNA Mismatch Repair Deficiency in Inflammatory Bowel Disease Associated Colorectal Carcinomas Jeremy S. Ditelberg, Shelley Karl, Mark Redston Background: Colorectal cancer (CRC) occurring in the setting of Inflammatory Bowel Disease (IBD) is relatively uncommon. Compared to sporadic CRC, the molecular pathogenesis of IBD-associated CRC has some distinctive features, particularly the timing of acquisition of genetic changes during progression through high grade dysplasia. DNA mismatch repair (MMR) deficient sporadic CRC is a well-defined entity comprised of Lynch syndromeassociated cancers and cancers characterized by a CpG island methylator phenotype (CIMP). The latter cancers characteristically arise in sessile serrated adenomas/polyps. The role of MMR deficiency in IBD CRC is less well established. We undertook this study to determine the frequency and clinical pathologic associations of MMR deficiency in IBD-associated neoplasia. Methods: Patients with CRC were selected from the pathology database of Miraca Life Sciences. The database includes demographic and clinical information, summary of the endoscopic report, biopsy location, and the histopathologic report for each biopsy specimen. Cases of CRC with a report date from 11/1/2007 to 11/30/2011 were extracted. From these cases, patients with a history of Ulcerative Colitis (UC) and Crohn Disease (CD) were extracted. Immunohistochemical (IHC) staining for MMR proteins MLH1, MSH2, MSH6 and PMS2 were performed on the CRC and interpreted as intact (positive staining) or deficient (loss of staining). Results: Biopsies from 917,599 colonoscopies were interpreted on 878,835 patients (51% female, 49% male) during the study period. Of these, 16,631 unique patients (48% female, 52% male) had histological and clinical features of UC while 5,736 patients had histological and clinical features of CD. During this period, 6,211 CRCs were diagnosed (52% male, 48% female). Of these CRCs, 22 occurred in patients with UC (prevalence of 22/16,631 = 0.13%), and 5 occurred in patients with CD (prevalence of 5/ 5,736 = 0.09%). 19 IBD patients were male (70.4%) and 8 were female (29.6%). The mean age was 58 years (median 56 years, standard deviation 13.5 years, range 33 to 84 years). Of these tumors, 14 (51.9%) were moderately differentiated (MD) and 13 (48.1%) were poorly differentiated (PD). IHC staining for MMR proteins showed MLH1 and PMS2 deficiency abnormalities in 3/27 (11%) patients, all of whom had UC. These 3 patients were 65, 77 and 79 years old and had CRC in the cecum, rectum and left colon, respectively. MSH2 and MSH6 expression were intact in all cases. Conclusions: MMR protein deficiency in the setting of IBD associated CRC appears to be less frequent than in the sporadic setting. Demographic characteristics suggest that it is likely due to MLH1 promoter methylation. Additional studies are needed to further define the mechanism. CRC occurring in the setting of IBD is rare, occurs predominantly in males and tends to be PD.
Mo1952 Matrix Metalloprotease (MMP9) Mediates Protection in Colitis Associated Cancer (CAC) by Modulating Cell Cycle Arrest via p21WAF1 Lewins Walter, Vaishali Pannu, Ritu Aneja, Pallavi Garg Background and Aim: Individuals with IBD have increased risk of developing colitis associated cancer (CAC). However, the pathogenesis of CAC is incompletely understood. Matrix metalloproteinases (MMPs) are zinc-dependent neutral endopeptidases that participate in degradation of extra cellular matrix proteins that occurs in both normal tissue remodeling and a variety of pathological processes including inflammation and cancer. MMP9 protein expression and activity is undetectable in normal tissues but is highly expressed in inflammatory state. We have shown that despite being a mediator of inflammation epithelial cell derived MMP9 plays a protective role in the development of CAC. Aim of this study is to comprehend the mechanism by which MMP9 mediates protection in CAC. Methods: MMP9-/- and wild type mice (WT) with and without CAC were used as in vivo model. Stably transfected HCT116 cells with pEGFP plasmid with & without MMP9 and mouse embryonic fibroblast cells (MEFs) obtained from MMP9-/- and WTs were used as in vitro models. Cell cycle events were analyzed by flow cytometry. Immunostaining was performed to detect DNA damage. Results: MMP9-/- mice exhibited significantly decreased expression of p53 and p21WAF1/ Cip1 compared to WTs in CAC. HCT116 cells overexpressing MMP9 showed a significant decrease in proliferation at 48 and 72 hours compared to vector. There was significant increase in p53 (8±0.32 fold) and p21WAFI/Cip1 (15±0.86 fold) expressions but a significant decrease in cyclinD1 expression (45±2.34 percent) in MMP9 overexpressing cells compared to vector. Overexpressing MMP9 cells exhibited a significant decrease in cyclinA expression (30±8.5 percent) but there was no significant change in cyclin E expression compared to vector. Results obtained from MEFs supported these findings, as irradiated MMP9-/- MEFs have significant decrease in p53 and p21WAF1/Cip1 expressions compared to WT MEFs. MMP9-/- MEFs also indicated a significant increase in cyclinA (3±0.5 fold) protein levels with subtle difference in cyclinE protein levels. Flow cytometry of MMP9 overexpressing cells showed a significant arrest of 8.5% in S phase of cell cycle compared to vector. DNA double strand breaks detected by immunostaining of H2AX showed less DNA damage in
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AGA Abstracts
AGA Abstracts
(p=0.03), and 5.3-fold in UCD (p=0.003) compared to normal controls. Compared to UC without dysplasia, miR-193a-3p was down-regulated 1.2-fold in UCrD (p=0.7) and 1.9-fold in UCD (p=0.05). UCD samples demonstrated increased methylation compared to normal controls and UC without neoplasia. There was a linear trend with decreased expression and increased methylation across all samples. 5-AZA treatment resulted in increased pri-miR193a transcript abundance in DLD1 (2.1-fold, p= 0.002), HCT116 cells (2.2-fold, p=0.02), and LoVo cells (1.2-fold, p=0.7), without significant change in mature miR-193a-3p expression. Transfection of 20nM and 50nM of miR-193a-3p in HCT116 cells down-regulated IL17RD, but not KRAS. Discussion: The gene encoding miR-193a-3p is methylated and its expression is down-regulated in IBD-associated neoplasia, suggesting that miR-193a-3p is regulated epigenetically. We identified IL17RD, an orphan receptor that potentiates IL-17 signaling, as a novel target of miR-193a-3p. As IL-17 signaling is up-regulated in inflammation and carcinogenesis, we postulate that down-regulation of miR-193a-3p contributes to cancer development in IBD via increased IL17RD.