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947 CpG Island Promoter Hypermethylation of RASSF1A Contributes Gastric Carcinogenesis
of adenoma from normal samples. Interestingly, 34 methylated and 47 demethylated CpG sites individually yielded 100% classification rates. CONCLUSIONS Genome-wide arraybased analysis of low-grade colorectal adenomas reveals extensive DNA methylation differences involving pathways associated with regulation of differentiation, apoptosis, and innate immunity. A number of differentially methylated sites appear to have the capacity to differentiate between normal and adenomatous tissue, and represent potential biomarkers for screening. Validation of these findings in a large sample would be desirable. 947 CpG Island Promoter Hypermethylation of RASSF1A Contributes Gastric Carcinogenesis Yong Jeoung, Moon Kyung Joo, Jong-Jae Park, Hyo Soon Yoo, Jung Wan Choe, Jiwon Kim, Ho Kim, Hyo jung Kim, Beom Jae Lee, Jae Seon Kim, Young-Tae Bak Among tumor suppressor genes located within 3p21.3 locus, Ras association domain family 1A gene (RASSF1A) has been extensively investigated because it has broad spectrum of effects on tumorigenesis, including cell proliferation and apoptosis. Methylation rates of RASSF1A were variously reported from 7.5% to 66.7% in gastric carcinoma tissues, and its functional roles in gastric carcinoma cells were not reported yet. We tried to reveal promoter hypermethylation of RASSF1A in both gastric adenocarcinoma tissues and gastric cancer cell lines, and investigate the effects of RASSF1A in gastric carcinoma cell lines. We found that gene expression of RASSF1A was nearly eliminated in SNU-719, MKN-28 and AGS cells, while it was observed in SNU-16 and KATO-III cells, and weakly positive in MKN45. By methylation specific PCR (MSP), we identified methylation-specific band only SNU719, MKN28 and AGS cells (full methylation), all of which did not show RASSF1A expression. In contrast, SNU-16, MKN-45 and KATO-III cells showed both methylation and unmethylation-specific bands (partial methylation), all of which had positive or weakly positive gene expression. Bisulfite sequencing in AGS and SNU-719 cells revealed that almost all CpG sites were densely methylated. When treating SNU-719, MKN-28 and AGS cells with demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dc), gene expression was restored and MSP pattern was altered in all three cell lines. Transfection of RASSF1A-expressing plasmid in AGS and SNU-719 cells significantly inhibited cell proliferation. Exogenous RASSF1A also reduced the expression of Cyclin D1 and phospho-pRb, and increased p27 by Western blot analysis. Furthermore, RASSF1A expression was significantly reduced (P=0.048) and methylation rate was elevated in gastric adenocarcinoma tissues, comparing with adjacent non-tumorous intestinal metaplasia (34.6% vs. 66.7%, P=0.029). Taken together, we conclude that epigenetic silencing of RASSF1A is frequently caused by promoter hypermethylation in both gastric cancer cell lines and gastric adenocarcinioma tissues, which contributes gastric carcinogenesis.
945 Promoter Hypermethylation of a Novel Tumor Suppressor Gene CA4 Associated With Colon Cancer Recurrence Jingwan Zhang, Xiaoxing Li, Jia Wang, Jing Gao, Minnie Y. Go, Joseph J. Y. Sung, Jun Yu BACKGROUND & AIMS: Using Methylated DNA Immunoprecipitation (MeDIP) combined with microarray assay, Carbonic Anhydrase IV (CA4) was identified to be preferentially methylated in colon cancer. CA4 is a member of carbonic anhydrases family which catalyzes the reversible hydration of carbondioxide. However, the functional role of CA4 in colon cancer is still unclear. METHODS: CA4 promoter methylation status was evaluated by bisulfite genomic sequencing. The biological functions of CA4 were determined in vitro cell viability, colony formation, cell apoptosis, and cell cycle assays and in vivo tumorigenicity assay. The co-operators of CA4 were identified by co-immunoprecipitation assay, mass spectrometry, promoter luciferase reporter assay and 2-dimensional gel electrophoresis assay. Clinical application of CA4 methylation was assessed in 115 primary colon cancer tissues. RESULT: We first screened the expression status of CA4 in 9 colon cancer cell lines and primary colon cancer tissues. CA4 was silenced in all 9 cell lines and the mean protein expression level of CA4 was significantly lower in colon cancer tissues as compared to their adjacent normal tissues (P<0.01). Silence of CA4 was mediated by promoter methylation. Re-expression of CA4 in colon cancer cell lines (Caco2, DLD-1, HCT116, HT29, LS180, SW1116) suppressed cell viability (P<0.001) and colony formation (P<0.01), and caused cell cycle arrest in G1 phase (P<0.05). CA4 also induced cell apoptosis (P<0.01), concomitant with enhanced protein expression of pro-apoptotic factors cleaved caspase-3, caspase-7, caspase-8 and PARP. The in vivo growth of colon cancer cells in nude mice was also markedly inhibited by stable transfection with vector harboring CA4 (P<0.05). We further tested the molecular mechanisms of CA4 in colon cancer as a tumor suppressor. Co-immunoprecipitation assay revealed that WTAP, a negative regulator in Wnt pathway, is directly interacted with CA4. Transfection of CA4 in colon cancer cell lines suppressed WTAP protein expression. Luciferase reporter assay showed that CA4 significantly reduced the activity of Wnt pathway (P<0.05), which was confirmed by down-regulation of active β-catenin and c-myc proteins. Whist, knock-down CA4 abolished the effect of CA4 on blocking Wnt signaling. Moreover, CA4 hypermethylation was detected in 75.7% (87/115) of colon cancer patients. Multivariate analysis and recurrence curve revealed that patients with CA4 methylation had significantly higher opportunities to suffer from cancer recurrence (P<0.05). CONCLUSIONS: Epigenetic inactivation of CA4 is a common event in colon cancer which may play a pivotal role as a novel tumor suppressor in colon carcinogenesis. CA4 directly interacts with WTAP to block Wnt signaling pathway. Detection of CA4 methylation may serve as an independent biomarker for the recurrence of colon cancer.
948 Imipramine for Treatment of Esophageal Hypersensitivity and Functional Heartburn: a Randomized Double-Blind, Placebo-Controlled Trial Julajak Limsrivilai, Phunchai Charatcharoenwitthaya, Nonthalee Pausawasdi, Somchai Leelakusolvong Background/Aims: Functional esophageal disorder is a common clinical dilemma that can diminish patient quality of life (QoL). Antidepressants show some benefits in patients with functional gastrointestinal disorders. This study aimed to assess the effectiveness of imipramine in esophageal hypersensitivity or functional heartburn. Methods: Patients with persistent reflux symptoms who failed to respond to proton-pump inhibitors were enrolled in a randomized, double blind, placebo-controlled trial. A total of 124 participants underwent upper gastrointestinal endoscopy and 24 h impedance-pH monitoring. Based on the results of these studies, patients diagnosed with esophageal hypersensitivity or functional heartburn were recruited and randomly assigned to receive 8 weeks of imipramine 25 mg or placebo before bed time. The reflux symptoms and QoL were assessed before and at 4 and 8 weeks after intervention using gastroesophageal reflux disease (GERD) and SF-36 score, respectively. The primary outcome was overall treatment response, defined as a greater than 50% decrease of GERD score after 8 weeks of treatment. Secondary outcome was the effect on QoL. Results: Eighty three patients were recruited and 80% were females with a mean age of 49 years (range 24-80). The imipramine (n=43) and placebo (n=40) groups were balanced at entry with respect to age, sex, body mass index, a history of alcohol use, smoking and coffee consumption, GERD score and duration of symptoms. Sixty seven patients completed the study (33 with imipramine and 34 with placebo); 8 discontinued medication due to mild adverse events (imipramine group, 4 dizziness, 1 palpitation and 1 difficult urination; placebo group, 1 dizziness and 1 eye pain); and 8 patients did not adhere to the protocol. There was no significant difference in response rates with imipramine compared to placebo (intention-totreat [ITT]; 37.2% vs. 37.5%, odds ratio [OR], 0.99; 95% confidence interval [CI] 0.412.41 and per-protocol [PP]; 45.5% vs. 41.2%, OR, 1.19; 95% CI, 0.45-3.13). Furthermore, subgroup analysis showed that neither esophageal hypersensitivity nor functional heartburn patients responded to treatment. Nonetheless, imipramine was associated with significant improvement of QoL (total SF-36 score, 72±17 and 61±19, p=0.048; physical functioning domain, 78±23 and 68±22, p=0.03; and emotion domain, 82±31 and 54±42, p=0.02) on PP analysis. However, ITT analysis did not reveal any differences between imipramine and placebo (total SF-36 score, 68±19 and 61±19, p=0.26). The side effects were not different between both groups except constipation that was more common with imipramine (51.2% vs. 22.5%, p=0.01). Conclusion: While low dose imipramine shows potential benefits on QoL, it is not more effective than placebo in terms of symptoms relief in patients with esophageal hypersensitivity or functional heartburn.
946 Genome-Wide Analysis of DNA Methylation in Low-Grade Colorectal Adenomas and Normal Colonic Mucosa Paul J. Lochhead, Claus-Dieter Mayer, Georgina L. Hold, Emad El-Omar BACKGROUND Aberrant methylation and demethylation of CpG sites, in concert with genetic alterations, may be important in the initiation and progression of colorectal neoplasia. The ability to detect methylated DNA in stool has generated interest in methylation biomarkers as potential screening tools for the detection of adenomas and colorectal cancer. Most colorectal neoplasia-associated methylation markers have been identified from studies on established carcinoma. Analyses of DNA methylation in benign adenomas have tended to focus on limited numbers of target sites, and few studies have applied genome-wide approaches to the evaluation of DNA methylation in colorectal premalignant lesions. We therefore set out to assess differences in DNA methylation between low-grade colorectal adenomas and paired normal mucosal specimens using genome-wide array-based methodology. METHODS A total of 23 paired adenoma and normal mucosal fresh tissues specimens were obtained from individuals undergoing screening colonoscopy. Adenomatous mucosa was sampled from polypectomy specimens, later confirmed histologically to be adenomas with low-grade epithelial dysplasia. Normal colonic mucosa was obtained from endoscopic biopsies taken from the same anatomic segment as the polypectomy. Bisulfite converted DNA from adenoma/normal pairs was analysed using the Illumina Infinium HumanMethylation platform, with 11 sample pairs run on 27K arrays, and the remaining 12 pairs run on 450K arrays. Array data were analysed using the R software environment. RESULTS Compared to normal mucosa, the most highly methylated genes in adenomas included the basic helixloop-helix transcription factor TWIST1, and the zinc finger protein ZNF625. The most highly demethylated genes in adenomas included tensin-4 (TNS4, a paralog of PTEN), and myoglobin (MB). Agglomerative hierarchical clustering on both 27K and 450K datasets demonstrated perfect clustering of normal and adenoma samples. Pathways analysis revealed methylation changes involving numerous pathways, including defensin, and netrin/DCC-mediated pathways. Using data from the 27K array as a training set, and the 450K array as a test set, we evaluated the minimum number of classifiers required to achieve complete discrimination
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AGA Abstracts
AGA Abstracts
significantly associated with age at diagnosis, age at surgery and disease duration in the rectal tissues. In contrast, no associations were observed between miR-34b/c methylation levels and any of the clinicopathological factors. Methylation levels of all 5 miRNAs were significantly higher in neoplasia (dysplasia and cancer) compared to non-neoplastic tissues. In addition, the methylation levels of all 5 miRNAs in rectal specimens were significantly higher in patients with cancer than in those without it, suggesting the presence of a "field defect" in the rectal tissues of UC patients. Receiver operating characteristics (ROC) analysis revealed that methylation levels of all 5 miRNAs in non-neoplastic rectal tissues successfully discriminated between patients with cancer from those without it. More importantly, the methylation status of miR-137 in non-neoplastic rectal tissues distinguished UC patients with and without neoplasia (AUC = 0.77), and emerged as an independent predictor of neoplasia (OR: 5.66; 95% CI: 1.37-23.47; P = 0.0168). Conclusions: The methylation levels of miR-1,-9, -124 and -137 displayed an age- and cancer-related pattern and indicated existence of a "field defect" in patients with UC. Methylation of these miRNAs in rectal biopsies—particularly miR-137—may potentially be a clinically-relevant biomarker for identifying patients at high risk for developing ulcerative colitis.
945 Promoter Hypermethylation of a Novel Tumor Suppressor Gene CA4 Associated With Colon Cancer Recurrence Jingwan Zhang, Xiaoxing Li, Jia Wang, Jing Gao, Minnie Y. Go, Joseph J. Y. Sung, Jun Yu BACKGROUND & AIMS: Using Methylated DNA Immunoprecipitation (MeDIP) combined with microarray assay, Carbonic Anhydrase IV (CA4) was identified to be preferentially methylated in colon cancer. CA4 is a member of carbonic anhydrases family which catalyzes the reversible hydration of carbondioxide. However, the functional role of CA4 in colon cancer is still unclear. METHODS: CA4 promoter methylation status was evaluated by bisulfite genomic sequencing. The biological functions of CA4 were determined in vitro cell viability, colony formation, cell apoptosis, and cell cycle assays and in vivo tumorigenicity assay. The co-operators of CA4 were identified by co-immunoprecipitation assay, mass spectrometry, promoter luciferase reporter assay and 2-dimensional gel electrophoresis assay. Clinical application of CA4 methylation was assessed in 115 primary colon cancer tissues. RESULT: We first screened the expression status of CA4 in 9 colon cancer cell lines and primary colon cancer tissues. CA4 was silenced in all 9 cell lines and the mean protein expression level of CA4 was significantly lower in colon cancer tissues as compared to their adjacent normal tissues (P<0.01). Silence of CA4 was mediated by promoter methylation. Re-expression of CA4 in colon cancer cell lines (Caco2, DLD-1, HCT116, HT29, LS180, SW1116) suppressed cell viability (P<0.001) and colony formation (P<0.01), and caused cell cycle arrest in G1 phase (P<0.05). CA4 also induced cell apoptosis (P<0.01), concomitant with enhanced protein expression of pro-apoptotic factors cleaved caspase-3, caspase-7, caspase-8 and PARP. The in vivo growth of colon cancer cells in nude mice was also markedly inhibited by stable transfection with vector harboring CA4 (P<0.05). We further tested the molecular mechanisms of CA4 in colon cancer as a tumor suppressor. Co-immunoprecipitation assay revealed that WTAP, a negative regulator in Wnt pathway, is directly interacted with CA4. Transfection of CA4 in colon cancer cell lines suppressed WTAP protein expression. Luciferase reporter assay showed that CA4 significantly reduced the activity of Wnt pathway (P<0.05), which was confirmed by down-regulation of active β-catenin and c-myc proteins. Whist, knock-down CA4 abolished the effect of CA4 on blocking Wnt signaling. Moreover, CA4 hypermethylation was detected in 75.7% (87/115) of colon cancer patients. Multivariate analysis and recurrence curve revealed that patients with CA4 methylation had significantly higher opportunities to suffer from cancer recurrence (P<0.05). CONCLUSIONS: Epigenetic inactivation of CA4 is a common event in colon cancer which may play a pivotal role as a novel tumor suppressor in colon carcinogenesis. CA4 directly interacts with WTAP to block Wnt signaling pathway. Detection of CA4 methylation may serve as an independent biomarker for the recurrence of colon cancer.
948 Imipramine for Treatment of Esophageal Hypersensitivity and Functional Heartburn: a Randomized Double-Blind, Placebo-Controlled Trial Julajak Limsrivilai, Phunchai Charatcharoenwitthaya, Nonthalee Pausawasdi, Somchai Leelakusolvong Background/Aims: Functional esophageal disorder is a common clinical dilemma that can diminish patient quality of life (QoL). Antidepressants show some benefits in patients with functional gastrointestinal disorders. This study aimed to assess the effectiveness of imipramine in esophageal hypersensitivity or functional heartburn. Methods: Patients with persistent reflux symptoms who failed to respond to proton-pump inhibitors were enrolled in a randomized, double blind, placebo-controlled trial. A total of 124 participants underwent upper gastrointestinal endoscopy and 24 h impedance-pH monitoring. Based on the results of these studies, patients diagnosed with esophageal hypersensitivity or functional heartburn were recruited and randomly assigned to receive 8 weeks of imipramine 25 mg or placebo before bed time. The reflux symptoms and QoL were assessed before and at 4 and 8 weeks after intervention using gastroesophageal reflux disease (GERD) and SF-36 score, respectively. The primary outcome was overall treatment response, defined as a greater than 50% decrease of GERD score after 8 weeks of treatment. Secondary outcome was the effect on QoL. Results: Eighty three patients were recruited and 80% were females with a mean age of 49 years (range 24-80). The imipramine (n=43) and placebo (n=40) groups were balanced at entry with respect to age, sex, body mass index, a history of alcohol use, smoking and coffee consumption, GERD score and duration of symptoms. Sixty seven patients completed the study (33 with imipramine and 34 with placebo); 8 discontinued medication due to mild adverse events (imipramine group, 4 dizziness, 1 palpitation and 1 difficult urination; placebo group, 1 dizziness and 1 eye pain); and 8 patients did not adhere to the protocol. There was no significant difference in response rates with imipramine compared to placebo (intention-totreat [ITT]; 37.2% vs. 37.5%, odds ratio [OR], 0.99; 95% confidence interval [CI] 0.412.41 and per-protocol [PP]; 45.5% vs. 41.2%, OR, 1.19; 95% CI, 0.45-3.13). Furthermore, subgroup analysis showed that neither esophageal hypersensitivity nor functional heartburn patients responded to treatment. Nonetheless, imipramine was associated with significant improvement of QoL (total SF-36 score, 72±17 and 61±19, p=0.048; physical functioning domain, 78±23 and 68±22, p=0.03; and emotion domain, 82±31 and 54±42, p=0.02) on PP analysis. However, ITT analysis did not reveal any differences between imipramine and placebo (total SF-36 score, 68±19 and 61±19, p=0.26). The side effects were not different between both groups except constipation that was more common with imipramine (51.2% vs. 22.5%, p=0.01). Conclusion: While low dose imipramine shows potential benefits on QoL, it is not more effective than placebo in terms of symptoms relief in patients with esophageal hypersensitivity or functional heartburn.
946 Genome-Wide Analysis of DNA Methylation in Low-Grade Colorectal Adenomas and Normal Colonic Mucosa Paul J. Lochhead, Claus-Dieter Mayer, Georgina L. Hold, Emad El-Omar BACKGROUND Aberrant methylation and demethylation of CpG sites, in concert with genetic alterations, may be important in the initiation and progression of colorectal neoplasia. The ability to detect methylated DNA in stool has generated interest in methylation biomarkers as potential screening tools for the detection of adenomas and colorectal cancer. Most colorectal neoplasia-associated methylation markers have been identified from studies on established carcinoma. Analyses of DNA methylation in benign adenomas have tended to focus on limited numbers of target sites, and few studies have applied genome-wide approaches to the evaluation of DNA methylation in colorectal premalignant lesions. We therefore set out to assess differences in DNA methylation between low-grade colorectal adenomas and paired normal mucosal specimens using genome-wide array-based methodology. METHODS A total of 23 paired adenoma and normal mucosal fresh tissues specimens were obtained from individuals undergoing screening colonoscopy. Adenomatous mucosa was sampled from polypectomy specimens, later confirmed histologically to be adenomas with low-grade epithelial dysplasia. Normal colonic mucosa was obtained from endoscopic biopsies taken from the same anatomic segment as the polypectomy. Bisulfite converted DNA from adenoma/normal pairs was analysed using the Illumina Infinium HumanMethylation platform, with 11 sample pairs run on 27K arrays, and the remaining 12 pairs run on 450K arrays. Array data were analysed using the R software environment. RESULTS Compared to normal mucosa, the most highly methylated genes in adenomas included the basic helixloop-helix transcription factor TWIST1, and the zinc finger protein ZNF625. The most highly demethylated genes in adenomas included tensin-4 (TNS4, a paralog of PTEN), and myoglobin (MB). Agglomerative hierarchical clustering on both 27K and 450K datasets demonstrated perfect clustering of normal and adenoma samples. Pathways analysis revealed methylation changes involving numerous pathways, including defensin, and netrin/DCC-mediated pathways. Using data from the 27K array as a training set, and the 450K array as a test set, we evaluated the minimum number of classifiers required to achieve complete discrimination
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AGA Abstracts
AGA Abstracts
significantly associated with age at diagnosis, age at surgery and disease duration in the rectal tissues. In contrast, no associations were observed between miR-34b/c methylation levels and any of the clinicopathological factors. Methylation levels of all 5 miRNAs were significantly higher in neoplasia (dysplasia and cancer) compared to non-neoplastic tissues. In addition, the methylation levels of all 5 miRNAs in rectal specimens were significantly higher in patients with cancer than in those without it, suggesting the presence of a "field defect" in the rectal tissues of UC patients. Receiver operating characteristics (ROC) analysis revealed that methylation levels of all 5 miRNAs in non-neoplastic rectal tissues successfully discriminated between patients with cancer from those without it. More importantly, the methylation status of miR-137 in non-neoplastic rectal tissues distinguished UC patients with and without neoplasia (AUC = 0.77), and emerged as an independent predictor of neoplasia (OR: 5.66; 95% CI: 1.37-23.47; P = 0.0168). Conclusions: The methylation levels of miR-1,-9, -124 and -137 displayed an age- and cancer-related pattern and indicated existence of a "field defect" in patients with UC. Methylation of these miRNAs in rectal biopsies—particularly miR-137—may potentially be a clinically-relevant biomarker for identifying patients at high risk for developing ulcerative colitis.