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sMo1346

Prediction of Retained Common Bile Duct Stones (RCBDS) in Acute BiliaryPancreatitis: Evaluation of Laboratory Testing According to TimeJean-Maxime Picard, Jean-Daniel Baillargeon, Marie-Pierre Garant

Acute biliary pancreatitis (ABP) is caused by common bile duct stones (CBDS) migration.In the majority of cases, the CBDS will pass spontaneously in the duodenum, but in some,could be retained and may lead to complications. Many studies evaluated pancreatic andliver enzymes as non-invasive predictors for rCBDS. These prior studies used variable timingfor enzymes dosage and only few evaluated laboratory trends as predictors. Knowing thatCBDS migration is a dynamic process, time should be considered in the interpretation ofthese tests. The aims of this study were to 1) identify the best timing for dosage of enzymesto predict rCBDS and 2) evaluate if trends of enzymes improve the prediction of rCBDS.Methods: A retrospective chart review was done for all patients with ABP who underwentan endoscopic retrograde cholangiography (ERCP), endoscopic ultrasound (EUS), magneticresonance cholangiography (MRCP) or intraoperative cholangiogram (IOC) for suspicion ofrCBDS in our institution between January 2000 and December 2011. Retained CBDS wasdiagnosed if confirmed at ERCP, EUS, MRCP or IOC. Bilirubin, AST, ALT, alkaline phospha-tase (ALP), GGT, amylase and lipase done at admission and during the 48h preceding theconfirming test were collected. Blood samples done at 6:00 am (±6h) 2 days before, 1 daybefore and the day of ERCP/EUS/MRCP/IOC were identified respectively as T-48, T-24 andT0. Mann-Whitney was used for statistical comparison of median values and laboratorytrends. Univariate analysis was performed. Results: One hundred and fifty patients wereincluded, among which 41 had rCBDS. Median amylase at admission was significantly higherin patient with than without rCBDS (1970 vs 873 IU/L; p<0.05) but not at other times.Median ALP was significantly higher in patients with rCBDS at T-24 only (166 vs 127 IU;p<0.05). Median bilirubin was significantly higher in patients with rCBDS only at T-24 (24vs 14 μmol/L; p<0.05) and T0 (24 vs 12 μmol/L; p=0.001). Median AST and median GGTwere significantly higher in patients with rCBDS only at T0 (62 vs 36 IU/L; p<0.05 and619 vs 191; p<0.05). Trends for each enzyme at different timing were evaluated to predictrCBDS, but none of them showed any specific pattern that could be predictive. Conclusion:This study evaluates laboratory tests in the prediction of rCBDS according to time. Wefound that trends of enzymes do not predict rCBDS. Predictors for rCBDS were amylase atadmission, ALP at T-24, bilirubin at T-24 and T0, AST at T0 and GGT at T0. Theseobservations suggest that timing should be considered in the interpretation of biochemicaltests for suspected rCBDS in ABP.

Mo1645

P62/Sequestosome 1 Is an Effector of Chemotherapy-Induced Apoptosis inColorectal Cancer Cells With Disabled AutophagyKoichi Okamoto, Shengbing Huang, Chunrong Yu, Frank A. Sinicrope

Background. Autophagy is a lysosomal degradation process that is activated in cancer cellsto maintain survival under stressful stimuli. Autophagy inhibition can promote tumor cellapoptosis, however, their functional relationship is unclear. A potential link is p62/sequesto-some 1 (Huang, et al, J Biol Chem 286:40002-12, 2011), a multi-functional protein thatshuttles ubiquitinated proteins to the lysosome during autophagy and is up-regulated byautophagy inhibition. We determined whether p62 can mediate chemotherapy-inducedapoptosis. Methods. Autophagy (GFP-LC3 localization by confocal microscopy) andapoptosis (annexin V, caspase activation by immunoblotting/luminescence) were analyzedin human colorectal cancer (CRC) cell lines (HCT116, DLD1). Cells were incubated withABT-263, CPT-11, or TRAIL alone or combined with autophagy inhibitors. Caspase-8-deficient Jurkat or siRNA knockdown CRC cells were utilized. p62 mutants (PB1, UBA orLC3 binding region) were generated by site-directed mutagenesis. Protein-protein interactionwas analyzed by immunoprecipitation. Caspase-8 self-aggregation and colocalization werestudied in cells with endogenous p62 or LC3, or in cells with ectopic lentiviral mCherry-p62 or LC3 by transfecting a non-cleavable caspase-8 fused with fluorescent Venus proteins.Results. Suppression of endogenous p62 was shown to attenuate apoptosis induction byABT-263 (small molecule Bcl-2/Bcl-xL antagonist) or CPT-11. Conversely, ectopic p62 orits up-regulation by autophagy inhibition enhanced apoptosis by these drugs that wasassociated with caspase-8 activation, was BAX-dependent, and resulted from mitochondrialamplification. Dependence on caspase-8 was confirmed using caspase-8-deficient cells or bycaspase-8 siRNA. A direct activator of caspase-8, i.e., TRAIL (TNF-related apoptosis inducingligand) alone or with ABT-263, induced caspase-8 aggregation and co-localization with p62in association with a synergistic drug interaction. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 or CPT-11 + an autophagy inhibitor. Compared to wild typep62, p62 mutants with disabled apoptosis failed to co-localize with caspase-8 or to promoteits self-aggregation in ABT-263-treated cells. Caspase-8 interacted and co-localized withLC3II (autophagosome marker). p62 knockdown attenuated binding between caspase-8 andLC3II; p62 overexpression enhanced co-localization. LC3 knockdown did not affect thecaspase-8 and p62 interaction, suggesting that p62 facilitates caspase-8 translocation tothe autophagosome. Conclusion. In cells with disabled autophagy, p62 is an effector ofchemotherapy-induced apoptosis that enables caspase-8 activation on the autophagosome.These data suggest that up-regulating p62 may enhance chemosensitivity, and supportcurrent efforts to inhibit autophagy to enhance chemotherapeutic efficacy.

Mo1646

REC8, a Novel EBV-Associated Hypermethylated Gene, Contributes to thePathogenesis of EBV-Associated Gastric CancerJia Wang, Yao Zeng, Junhong Zhao, Jing Gao, Joseph J. Y. Sung, Jun Yu

aimed to analyses the epigenetic alteration and biological function of REC8 in EBV-associatedgastric cancer. Methods: Promoter methylation was examined by combined bisulphate restric-tion analysis and bisulfite genomic sequencing (BGS). The biological functions of REC8were evaluated by loss- and gain-of-function assays in vitro including MTT cell viabilityassay, colony formation, cell cycle analysis, Annexin V apoptosis assay and wound healing

S-626AGA Abstracts

assay, and in vivo tumorigenicity assalysis in nude mice. The molecular mechanism of REC8in gastric cancer was explored by human cancer pathway PCR array, and confirmed bywestern blot. Results: REC8 was significantly down-regulated in human primary gastriccancer tissues as compared with their adjacent non-tumor tissues (P=0.0007). REC8 wasalso down-regulated or silenced in 9 of 14 human gastric cancer cell lines (64.3%), andthis down-regulation was associated with its promoter methylation of REC8 gene. Themethylation level of REC8 was significantly higher in EBV-positive primary gastric cancertissues compared with EBV-negative gastric cancer tissues by BGS (P<0.0001). Promoterhypermethylation of REC8 was detected in EBV-positive gastric cancer cell line, AGS-EBV,with REC8 transcriptional silence, but not in EBV-negative gastric cancer cell line, AGS,with REC8 expression. Expression of REC8 was restored in AGS-EBV by exposure todemethylation agent. Ectopic expression of REC8 in gastric cancer cell lines (AGS-EBV andBGC823) significantly suppressed cell viability (P<0.01), colony formation (P<0.05), G1 toS phase transition (P<0.05), and cell migration ability (P<0.05), but induced cell apoptosis(P<0.05). In contrast, Knock-down of REC8 in normal gastric epithelial cell line GES1increased cell proliferation (P<0.01) and migration ability (P<0.01), but alleviated cellapoptosis (P<0.05). Moreover, re-expression of REC8 in AGS-EBV cells reduced tumor sizein nude mice (P<0.05). The molecular mechanism of REC8 as a tumor suppressor wasassociated with up-regulation of pro-apoptotic regulators (cleaved caspase3, caspase7 andPARP), cyclin-dependent kinase inhibitors (p21 and p27), and cell to cell adhesion gene(E-cadherin), while down-regulation of key cell proliferation regulators (Cyclin D1 andPCNA). Conclusions: REC8 is a novel methylated gene driven by EBV infection in gastriccancer. REC8 has an important tumor suppressive function in gastric cancer through modulat-ing cell proliferation, cell cycle, apoptosis, and migration. Epigenetic silencing of REC8by EBV infection may contribute to the pathogenesis of EBV-associated gastric cancer.Acknowledgment: This study was supported by research grant of RFCID Hong Kong(11100022).

Mo1647

Inhibition of EZH2/EZH1 by UNC1999 Enhances the Potency of the EGFRInhibitor Gefitinib in Colon Cancer CellsBryson Katona, Yuanyuan Liu, Anqi Ma, Jian Jin, Xianxin Hua

BACKGROUND: EGFR inhibitors have marginal efficacy in metastatic colon cancer, and inKRAS mutant tumors these inhibitors have been shown to decrease survival. As EGFR ishighly expressed in a subset of colon cancers and epigenetic regulation is often altered, anew approach to enhance the efficacy of therapy is to co-target both EGFR signaling andan epigenetic pathway. One targetable epigenetic regulator that is often over-expressed incolon cancer is the histone methyltransferase EZH2. EZH2 is a component of the polycombrepressive complex 2 (PRC2) and it catalyzes H3K27-methylation, ultimately leading to genesilencing of tumor suppressors. Inhibition of EZH2 activity has been shown to decreasecolon cancer cell proliferation, however the small molecule EZH2/EZH1 inhibitor UNC1999and the combination of EZH2/EZH1-EGFR co-inhibition have not yet been examined incolon cancer cells. AIMS: The aim of this study is to determine if EZH2/EZH1 inhibitionby UNC1999 affects both colon cancer cell growth and the response of colon cancer cellsto EGFR inhibition. METHODS: KRAS wild type HT-29 and KRAS mutant HCT-15 coloncancer cell lines were utilized. Cell proliferation was assessed via the MTS assay and cellcounting. Protein and mRNA levels were assessed via Western blotting and qPCR, respec-tively. EGFR was inhibited with the small molecule inhibitor gefitinib. Both EZH2 and EZH1were inhibited by the small molecule UNC1999. EZH2 was knocked down with lentiviraldelivered shRNAs. RESULTS: Treatment of HT-29 and HCT-15 cells with UNC1999 led toa dose dependent decrease in H3K27-methylation and cell proliferation. UNC1999 alonealso induced apoptosis in both cell lines at high concentrations. Co-treatment of HT-29 andHCT-15 cells with UNC1999 and gefitinib effectively inhibited H3K27-methylation andEGFR phosphorylation. This combination of UNC1999 and gefitinib led to a significantdecrease in cell proliferation and an increase in apoptosis induction when compared toeither compound alone. To determine if these effects were related to the EZH2 protein level,multiple EZH2 shRNAs were used to successfully knockdown EZH2 in both HT-29 andHCT-15 cells. Although EZH2 knockdown led to a decrease in H3K27-methylation, EZH2knockdown alone did not consistently decrease cell proliferation, induce apoptosis, noraugment the effects of gefitnib, indicating that UNC1999 effectiveness may not be relatedto total EZH2 levels and that EZH1 inhibition may also be critical for EGFR inhibitorenhancement. CONCLUSIONS: The EZH2/EZH1 inhibitor UNC1999 effectively suppressedH3K27-methylation in HT-29 and HCT-15 cells and demonstrated that EZH2/EZH1 inhibi-tion may serve as an effective method to increase the efficacy of EGFR inhibitors in coloncancer.

Mo1648

Enhancing the Efficacy of EGFR Inhibitors in Colon Cancer Cells ThroughTargeted Disruption of Menin-MLL1Bryson Katona, Leslee Sholomskas, Xianxin Hua

BACKGROUND: A new approach to enhancing the efficacy of chemotherapeutic regimensinvolves simultaneously co-targeting cell signaling and epigenetic mechanisms. Given theirmarginal efficacy in metastatic colon cancer, EGFR inhibitors are one class of chemotherapeu-tic agents that have the potential to be enhanced through targeted epigenetic modulation.In colon cancer, one epigenetic complex that remains poorly studied is menin and itshistone methyltransferase binding partner MLL1. In MLL-fusion leukemias, menin-mediatedrecruitment of MLL1 leads to enhanced H3K4-methylation resulting in increased expressionof Hox genes and increased proliferation. AIMS: The aim of this study is to define the roleof menin-MLL1 in cell proliferation and response to EGFR inhibitors in HT-29 colon cancercells. METHODS: Menin and MLL1 were knocked down with lentiviral delivered shRNAs.Cell proliferation was assessed via the MTS assay and cell counting. Protein and mRNAlevels were assessed via Western blotting and qPCR, respectively. EGFR was inhibitedwith the small molecule inhibitors gefitinib or erlotinib. MI-2-2, a menin inhibitor, wasindependently synthesized and used as a small molecule inhibitor of the menin-MLL1interaction. RESULTS: shRNA knockdown of MLL1 in HT-29 colon cancer cells decreased