AG

AA

bst

ract

scells. By transfection with miR-29c or after treatment with celecoxib, expression of Mcl-1,which is recently reported as one of the targets of miR-29 family (Oncogene 26: 6133,2007), is suppressed and apoptosis is induced in gastric cancer cells. Conclusions. SincemiR-29c suppresses Mcl-1 oncogene and is downregulated in early gastric cancers and gastricadenomas, miR-29c may play critical roles in the development of gastric tumors. Thesefindings suggest that COX-2 inhibition is effective in the chemoprevention of gastric cancervia the activation of miR-29c.

1089

Promoter Hypermethylation Mediates Downregulation of Thiamin ReceptorSLC19A3 But Not SLC19A2 in Gastric CancerXin Liu, Hongchuan Jin, Emily KY Lam, Xian Wang, Yun W Lam, Yuen Yee Cheng, JunYu, Francis K. L. Chan, Joseph J. Sung

Background: Gastric carcinogenesis is a multiple stage process resulting from the accumula-tion of oncogene activations and tumor suppressor gene inactivations. Tumor suppressorgenes can be inactivated through promoter hypermethylation in gastric carcinoma even pre-malignant gastric lesions. Therefore promoter hypermethylation was proposed as the markerto define novel TSGs and detect early gastric cancer. In an effort to search for such biomarkers,SLC19A3 (solute carrier family 19, member 3, or THTR2, thiamine transporter 2) was foundas a candidate TSG epigenetically downregulated in gastric cancer. Methods: SLC19A3expression was determined by RT-PCR. Methylation of SLC19A3 promoter was analyzedby methylation specific polymerase chain reaction (MSP) and bisulfite genome sequencing(BGS). The effect of SLC19A3 on cell growth was determined by colony formation assay.Results: The expression of SLC19A3 but not SLC19A2 (solute carrier family 19, member2, or THTR1, thiamine transporter 1), another member of thiamine transporter, was downreg-ulated in most of gastric cancer cell lines used (71%, 5/7). SLC19A3 donwregulation wassignificantly restored after the treatment of 5-aza-2'-deoxycytidine, a well-used demethylationagent, demonstrating that promoter methylation contributes to SLC19A3 silencing in gastriccancer cells. In addition, both MSP and BGS revealed that SLC19A3 promoter was methylatedin gastric cancer cell lines (67%, 4/6). Ectopic expression of SLC19A3 caused the significantgrowth inhibition of gastric cancer cells In Vitro (55%, p<0.05) suggesting that SLC19A3downregulation is important to gastric cancer development. Finally, methylation of SLC19A3promoter was also detected in primary gastric carcinoma tissues (47%, 47/100) as well asintestinal metaplasia tissues (32%, 8/25), indicating that methylation of SLC19A3 promoteris an early event during gastric carcinogenesis. Conclusions: In summary, SLC19A3 wasidentified as a novel candidate tumor suppressor gene downregulated through promoterhypermethylation in the development of gastric cancer. Promoter methylation of SLC19A3occurs at the early stage of gastric development and thus may be of value in monitoringgastric cancer development.

1090

The Aurora Kinase a Regulates GSK-3β/β-Catenin/Tcf Complex Signaling inGastric Cancer CellsAbbes Belkhiri, Altaf A. Dar, Mohammed Soutto, M. Blanca Piazuelo, Pelayo Correa, MaryKay Washington, Wael M. El-Rifai

Amplification at the 20q chromosomal region is a frequent finding in gastric cancer. In thisstudy, we have identified Aurora kinase A as a critical molecular target, at this region, thatis frequently amplified and over-expressed in gastric cancer. We detected relative DNA copynumber gains of AURKA in 40% of tumors (24 of 40 samples). We also detected mRNAover-expression in 60% of the tumors (35 of 60). Immunohistochemical analysis on tumortissue microarrays of gastric cancer confirmed the over-expression of AURKA in gastriccancer samples, as compared to normal gastric mucosa tissues. We further investigated therole of AURKA in regulating GSK-3β in gastric cancer cells. We first detected a significantincrease in the phosphorylation of GSK-3β at Ser 9 following the over-expression of AURKAin AGS cells. Using two-way co-immunoprecipitation assay for exogenous and endogenousconfirmed that GSK-3β and AURKA proteins coexist in the same protein complex. Usingan In Vitro kinase assay, we showed that the recombinant human AURKA protein phosphoryl-ated the GSK-3β protein at Ser 9 in a dose-dependent manner. Active GSK-3β is known tophosphorylate β-catenin and target it for proteasomal degradation; in line with our results,the increase in phospho-GSK-3β level was accompanied by a significant decrease in β-catenin phosphorylation (Ser33/37/Thr41) and nuclear accumulation of β-catenin protein.Furthermore, we have measured the β-catenin/TCF transcription activity using pTopFlashand its mutant pFopFlash luciferase reporter plasmids. Over-expression of AURKA signific-antly increased the pTopFlash activity, whereas the kinase dead AURKA mutant (D274A)had no effect. We corroborated this finding by detecting a significant mRNA up-regulationof several targets of the β-catenin/TCF transcription complex (Cyclin D1, c-MYC, c-MYC-binding protein, CLDN1, FGF18, and VEGF). As previous studies indicated that GSK-3βis an important downstream target of the PI3K/AKT survival pathway- and our earlier studyshowed that AURKA also phosphorylates AKT- we used AKT-specific inhibitor in the presenceand absence of AURKA to define the role of this direct interaction of AURKA and GSK-3β.Our results indicated high levels of phosphor-GSK-3β (Ser 9) in the presence of the inhibitorin AURKA over-expressing cells, confirming our finding that AURKA can directly bind andphosphorylate GSK-3β. Taken together, our results indicate that AURKA/GSK-3β axis is anovel mechanism for regulating the β-catenin/TCF transcriptional activity underscoring theoncogenic potential for AURKA in gastric tumorigenesis.

A-168AGA Abstracts

1091

Celecoxib Inhibits Apurinic/Apyrimidinic Endonuclease-1 Expression andPrevents Gastric Cancer in Helicobacter pylori-Infected Mongolian GerbilsSeiji Futagami, Tetsuro Kawagoe, Tomotaka Shindo, Akane Horie, Tatsuhiko Hamamoto,Masafumi Kusunoki, Kazumasa Miyake, Katya Gudis, Taku Tsukui, Sheila E. Crowe,Choitsu Sakamoto

Background&Aims: We have reported that celecoxib significantly prevented gastric cancerin H. pylori-infected Mongolian gerbils. Previous studies and our data have demonstratedthat H. pylori stimulation upregulates APE-1 (Apurinic/apyrimidinic endonuclease-1) expres-sion in gastric cancer cell lines and non-neoplastic gastric mucosal biopsies. The aim of thisstudy was to see whether celecoxib, a selective COX-2 inhibitor could prevent the develop-ment of gastric cancer via inhibition of H. pylori infection-induced APE-1 expression.Methods: 70 Mongolian gerbils were divided into six groups. Group 1 gerbils served ascontrol (n=6). 10 gerbils were given five biweekly MNU (N-Methyl-N-nitrosourea;30ppm)(Group 2). 6 short-term H. pylori-infected gerbils were sacrificed after 8 weeks H. pyloriinfection (Group 3). 6 long-term H. pylori-infected gerbils were sacrificed after 44 weeks H.pylori infection (Group 4). 20 gerbils were given five biweekly MNU pretreatment and long-term H. pylori infection (Group 5). In addition, after H. pylori inoculation, group 6 gerbilsalso received a 36-week administration of celecoxib in their diet. APE-1 expression aloneor with COX-2 in gastric tissues was evaluated by western blot and immunohistologicalanalysis. MPO activity and TBARS levels were also evaluated. Results: APE-1 was localizedin gastric epithelial cells and mesenchymal cells including macrophages in H. pylori-infectedgerbils. The number of APE-1 positive cells in group 4 and 5 gerbils were significantlyincreased compared to that of group 3 gerbils. MPO activity and TBARS levels in gerbilswith long-term H. pylori infection was significantly higher than in MNU-pretreated gerbils.MPO activity and TBARS levels in MNU-pretreated H. pylori-infected gerbils was significantlyhigher than in gerbils with long-term H. pylori infection. Celecoxib treatment significantlyreduced MPO activity, TBARS levels, and the incidence of gastric cancer. APE-1 and IkBαphsophorylation levels were significantly increased in MNU-pretreated H. pylori-infectedgerbils compared to those in MNU only gerbils. Celecoxib significantly reduced APE-1 andIkBα phsophorylation levels in MNU-pretreated H. pylori-infected gerbils. COX-2 and APE-1 were coexpressed in the macrophages in H. pylori-infected gerbils. Conclusion: Celecoxibprevented gastric cancer in MNU-pretreated H. pylori-infected gerbils with a reduction inAPE-1 expression thereby implicating APE-1 as playing a role in gastric carcinogenesis inthis model.

1092

Altered Macrophage Function Contributes to Colitis Development in PI3KP110δ Mutant MiceJennifer K. Uno, Kavitha N. Rao, Katsuyoshi Matsuoka, Shehzad Z. Sheikh, Fengling Li,Scott E. Plevy

Background: Complex interactions between the enteric microbiota and innate immune cellsare necessary to maintain gut homeostasis. Innate immune responses mediated by Toll-likereceptors (TLRs) while crucial for host defense against pathogens need to be tightly regulatedto prevent chronic inflammation in the inflammatory bowel diseases (IBD). Phosphoinositide-3 kinase (PI3K) is an important negative regulator of TLR signaling. Initial characterizationof mice with a targeted mutation in the p110δ subunit of PI3K revealed defects in B- andT-cell signaling as well as chronic, focal colitis. Aim: Here, we investigate innate immunedefects that may lead to the pathogenesis of colitis in these mice. Results: Spontaneouscolitis is demonstrated at 8 wks of age with disease severity increasing with age. A proinflam-matory mucosal and systemic cytokine profile was observed characterized by overexpressionof IL-12/23, TNF, IFN-γ and IL-17. Compared to wild type (WT) macrophages, PI3Kp110δ mutant macrophages demonstrate hyperresponsiveness to TLR ligands with increasedinflammatory cytokine expression. PI3K p110δ mutant macrophages also display defects inIL-10 and C5a mediated inhibition of IL-12 p40. In addition, PI3K p110δ mutant macro-phages were less efficient than WT macrophages in eradicating intracellular commensal K12E coli (n=4; p=0.05). Defective bacterial killing was also associated increased production of IL-12 p40 (n=3, p=0.05). Bactericidal activity was significantly augmented in WT macrophagespretreated with LPS and IFN-γ, but not in PI3K p110δ mutant macrophages (n=3, p=0.05).Increased expression of the autophagosomal marker LC3-II was observed in LPS-activatedWT macrophages, however PI3K mutant macrophages showed decreased basal and LPS-stimulated levels of LC3-II. Furthermore, LC3-II expression was increased in E coli infectedWT but not PI3K mutant macrophages, suggesting that alterations in the autophagocyticpathway may, in part, be responsible for decreased bactericidal activity. Furthermore, treat-ment with autophagy inducer rapamycin restored bactericidal activity in p110δ mutantmacrophages. Conclusion: This study confirms a critical role of PI3K p110δ in innateimmune responses against enteric microbes which could provide insight into the pathogenesisof human IBD.

1093

The Absence of Functional PI3Kγ Has a Protective Effect On DSS-InducedColitis in MiceWillemijn A. van Dop, Stefano Marengo, Anje A. Te Velde, Fiebo J. ten Kate, DanielHommes, Guy E. Boeckxstaens, Emilio Hirsch, Gijs R. van den Brink

Background & aims: Phosphatidylinositol-3-kinases (PI3Ks) play a crucial role at all stagesof immune-mediated diseases. PI3Kγ is a class 1b PI3K and is the major subclass of thePI3Ks that is activated through G-protein coupled receptors, in response to chemoattractants.It is expressed by leucocytes and endothelial cells and is responsible for the emigration ofleucocytes from the bloodstream to the site of injury or infection. In Ulcerative Colitisoveractivity of the immune system plays a major role. The constant influx of inflammatorycells to the affected tissue causes tissue damage. Reducing this influx, for example byinhibition of PI3Kγ, might therefore be a potential target for therapy. Here we investigatedthe role of PI3Kγ in a mouse-model of colitis. Methods: We used 10 mice with a point