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swere noted by six weeks. The results from the transcriptome and signaling pathway analysiswill be discussed. In summary, we have devised a model for sporadic colorectal cancer thatallows the study of tumor formation by mutated epithelial cells in the setting of normalstroma and permits longitudinal analysis by endoscopic modalities.

S2055

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 6 (CEACAM6) Is aPotential Biomarker for Peritoneal Metastasis of Gastric CancerLydia K. Lung, Angela M. Hui, Billy C. Leung, George G. Chen, Enders K. Ng

Background: Gastric adenocarcinoma has a strong predilection to peritoneal metastases.However, the underlying mechanism remains enigmatic. CEACAM6 is a glycosylphosphatidy-linositol (GPI)-linked molecule, which has been shown in recent studies to be frequentlyupregulated in a variety of cancers. This study aims to investigate the role of CEACAM6 asa potential biomarker and also a possible key mediating factor for peritoneal disseminationof stomach cancer. Methods and Results: Gastric cancer cell lines AGS, KatoIII, MKN28 andMKN45 were respectively co-cultured with human peritoneal mesothelial cell (HPMC)extracted from freshly excised omental tissue. CEACAM6 expression in the culture mediaas determined by Western blot was found to be upregulated by 79.33% in the co-culturesettings when compared to that with cancer cells alone. Following these preliminary findings,CEACAM6 was forcibly overexpressed in AGS, KatoIII and MKN28 but silenced in MKN45.Colony formation assay by seeding 5000 non-aggregated cells of the treated cell lines into6-well plates was performed. While forced expression of CEACAM6 in MKN28 led to asubstantial rise in colony formation ability (195%, p<0.0001), CEACAM6-silenced MKN45had a marked declination by 45.83% (P=0.0002). Though statistically insignificant, forciblyexpression of CEACAM6 in AGS and KatoIII had also yielded a surge in colony formationability by 106.9% and 100.2%, respectively. The influence of CEACAM6 towards cellularproliferation was further elucidated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS assay). Comparing to the wild types, CEA-CAM6 overexpressing AGS and MKN28 had resulted in a 203.95% (P=0.0002) and 56.37%(P=0.0026) increase in proliferative activity, respectively. To the contrary, CEACAM6-silenced MKN45 had a 40.27% (P=0.0255) reduction. The effect of CEACAM6 towards cellcycle was further elucidated using flow cytometry. 2x10e5 cells were harvested and stainedwith propidium iodide/Triton X-100 solution for analysis by 488-nm argon ion laser. Whileno significant variations were observed in other cell lines, a drastic declination of 90.48%of G2/M phase was observed in CEACAM6-silenced MKN45. Conclusions: Exposure ofgastric cancer cells to HPMC is a potent stimulus to upregulation of CEACAM6. Over-expression of CEACAM6 in turn enhances both colony formation ability and proliferationrate of gastric cancer cells In Vitro. These are possible facilitating factors for trans-celomicspread of stomach cancer.

S2056

Mucosally-Adherent E. coli Isolates from Human Colon Cancer Induce NuclearTranslocation of β-Catenin and COX-2-Dependent Cyclin D1 Expression inHuman Colon Cancer Epithelial CellsPaul D. Collins, Lu-Gang Yu, Jonathan M. Rhodes

INTRODUCTION: Dysregulation of Wnt/β-catenin signaling with nuclear translocation ofβ-catenin is an early event in colon cancer development[1]. E. coli are found in increasednumbers adherent to colonic mucosa in patients with colon cancer[2]. Bacterial involvementin cancer progression could explain the much higher rate of cancer in the colon than thesmall intestine. AIMS & METHODS: DLD-1 cells, a colon cancer cell line with biallelicinactivation of APC gene, were cultured with E. coli mucosal isolates from colon cancerpatients. Cells were incubated with Prostaglandin-E2 (0.1µM) as a positive control. Cellularlocalization of β-catenin was quantified using immunofluorescence microscopy and expressedas a ratio of the intensity of nuclear to whole cell fluorescence (N:WC). RESULTS: Six ofnine mucosal E. coli isolates from colon cancer induced nuclear translocation of β-catenin.E. coli isolate HM545 increased nuclear localization (mean ratio N:WC = 1.064; sd 0.406)compared to the negative control (No bacteria N:WC=0.982; sd=0.087) (p=0.017). NeitherHM545 supernatant (N:WC=0.983; sd=0.085) (p=0.90) nor heat-killed HM545 (N:WC=0.997; sd=0.117) (p=0.25) increased nuclear localization. The expression of Cyclin D1,known to be responsive to β-catenin nuclear localisation, increased markedly in responseto three of five E. coli isolates (including HM545), but not in response to HM545 supernatantor heat-killed bacteria or with the laboratory control E. coli K12 (Fig. 1). HM545-inducedexpression of Cyclin D1 was inhibited by the cox-2 inhibitor NS398 (10µM) and solubleplantain fibre (5mg/ml), an inhibitor of E. coli epithelial cell adhesion (Fig. 1). CONCLUSION:Mucosal E. coli isolates from colon cancer induce nuclear localization of β-catenin andactivation of Cyclin D1 in human colon cancer cells. This effect requires whole, live bacteriaand is cox-2 dependent. This is a plausible mechanism by which mucosa-associated bacteriamay promote the colon cancer development. REFERENCES: 1 Bienz et al. Cell. 2000;103:3112 Martin et al. Gastroenterology. 2004;127:80

Fig. 1 Epithelial cell Cyclin D1 expression in response to E. coli

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S2057

A Loss of Symbiosis with Certain Strains of Commensal E Coli Leads toIncreased Colon Cancer Invasion in An In Vitro Colon Cancer Model ViaActivation of Rac and MMP-1Sarah C. Glover, Marinka Bulic, Ramana V. Vishnubhotla, V. K. Viswanathan, MichaelCho, Jennifer L. Roxas, Rebecca Mecum, Igor Titushkin

Previous retrospective studies have shown that roughly 25% of colon cancer metastasizeregardless of stage. While numerous genetic and environmental contributors colon cancermetastasis have been identified, little has been done to address the role that luminal bacteriaplay in colon cancer invasion and spread. We have previously shown that the entericpathogen EPEC increases colon cancer invasion in an In Vitro model of colon cancer via itsability to modulate Rho and ROCK activities. In the course of performing these experimentswe discovered that certain commensal strains of E. coli also led to increased colon cancercell invasion in in virto models of colon cancer. However, commensal strains of E. coli didnot lead to substantial increases in Rho or ROCK activities. In order to determine themechanism through which commensal E. coli increased colon cancer invasion, we infectedboth NCM 460 (non-malignant) and Caco-2BBE (malignant) cells grown in 1.2 mg/ml 3-D type I collagen scaffolds with six stains of human commensal E. coli at an MOI=40 for 2hours followed by treatment of the cell containing scaffolds with 10 µg/ml gentamicin forat least 2 hours. At 24 hours post infection, we evaluated Rac-1 activity, depth of invasion,MMP-1 activity, and collagen degradation using standard molecular biology techniques andadvanced imaging. Intriguingly, 2 of the 6 strains of E. coli we evaluated led to a nearly 8-fold increase in Rac-1 activity over EPEC infection and nearly 16 fold over control butONLY in the Caco-2BBE containing scaffolds. Similarly, these same two commensal strainsproduced a nearly 4-fold increase in MMP-1 activity and a 10-fold increase in type 1 collagendegradation by Caco-2BBE cells but not by NCM 460 cells. Depth of invasion was unchangedin NCM 460 cells but was significantly increased over control in Caco-2BBE scaffolds infectedwith these two strains of commensal E. coli. Taken together, this data suggests that commensalbacteria can take on the role of pathogens in the setting of colon cancer leading to increasedcolon cancer invasion.

S2058

CagA C-Terminal Variations in Helicobacter pylori Strains from ColombianPatients with Gastric Precancerous LesionsLiviu A. Sicinschi, Pelayo Correa, Richard M. Peek, M. Constanza Camargo, M. BlancaPiazuelo, Judith Romero-Gallo, Uma S. Krishna, Alberto Delgado, Luis E. Bravo, BarbaraG. Schneider

Background: CagA is a bacterial effector which is disproportionately present among virulentHelicobacter pylori (H. pylori) strains. The C-terminus of CagA is polymorphic, bearing 3 ormore EPIYA sequences. Following the attachment of H. pylori to gastric epithelial cells, CagAis translocated into host cells where it undergoes tyrosine phosphorylation at the Tyr residuewithin the EPIYA-C motif. A prerequisite for CagA phosphorylation is multimerization,which is mediated by a conserved sequence of 16 amino acids, designated the CagA multimer-ization motif (CM). Because the number of EPIYA-C motifs is strongly related to the severityof precancerous lesions and gastric cancer (GC) in certain populations, we characterizedmolecular CagA variations in H. pylori strains isolated from Colombian patients from areasof high and low risk for GC. Materials and methods: Eighty adults with dyspeptic symptoms(39 from low- and 41 from high-risk areas) underwent gastrointestinal endoscopy. Gastricmucosal biopsies were obtained for histology and culture. Genomic DNA was isolated frombacteria cultured from antral biopsies from one strain per patient. The 3' region of thecagA gene was sequenced. Nucleotide sequence alignment, conceptual translation, andidentification of EPIYA motifs were performed using Geneious software. Results: Sixty-seven(83.8%) of 80 H. pylori strains were cagA positive. CagA proteins contained either three(64.2%), four (31.3%), or five EPIYA motifs (4.5%), all with Western type CagA-specificsequences. The number of EPIYA motifs was not associated with geographic region. However,strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinalmetaplasia and dysplasia), while strains with one EPIYA-C motif were associated with lesssevere gastric lesions (non-atrophic and multifocal atrophic gastritis) (P=0.001). In fifty-fiveof the strains, the CM motif was concordant with the EPIYA motif (Western type). Howeverin 12 strains, the CM motif varied and contained a peptide sequence previously seen inonly strains from East Asian countries. All 12 strains bearing the East Asian CM motifs wereisolated from subjects residing in the low risk for cancer region (P<0.001). Conclusions:The severity of precancerous lesions in Colombians was significantly associated with thenumber of CagA EPIYA-C motifs in infecting H. pylori strains. The reported difference inincidence of gastric cancer between the two areas may be related to the type of CagAmultimerization motif.

S2059

A 17-Year Delay of Development of Intestinal Type Adenocarcinoma inFemales Explains Male Predominance of Upper Gastrointestinal CancerMohammad H. Derakhshan, Sarah Liptrot, Douglas Morrison, Ian L. Brown, Kenneth E.McColl

Introduction We have recently demonstrated that the male predominance of upper GIadenocarcinoma is associated with the intestinal histological subtype and is independent ofanatomical location, i.e. oesophageal vs. gastric. We have undertaken kinetic analysis of agespecific incidence trends to determine the effect of the gender phenomenon. Methods Thestudy was based upon 812 cases of upper gastrointestinal adenocarcinoma randomly selectedfrom the West of Scotland cancer Registry between 1998 and 2002. The Lauren histologicsubtype of adenocarcinoma was determined by reviewing 1204 original reports and 3241biopsy and surgical slides. A regression curve modelling was used to calculate age-specificcurve equations of both diffuse and intestinal subtypes of cancer. Results The analysiscompromised 405 non-cardia cancer, 173 cardia cancer and 209 oesophageal adenocarcin-oma. The crude incidence rate of intestinal type upper GI adenocarcinoma was higher in