Tcf Signaling in Gastric Cancer

treatment strategy is best for the individual patient. The highly selective gastrin receptor antagonist YF476 has shown anti-tumour effects in various rodent models of gastric carcinogenesis driven by hypergastrinemia. YF476 is orally active and there have been no reports of significant adverse events in healthy volunteers. In the present study, we have examined the effect of YF476 in patients with gastric carcinoids type 1. Methods: Eight patients with gastric carcinoids type 1 have been included in a single centre, open-label, pilot, phase 2 trial of the gastrin receptor antagonist YF476. They were given YF476 by mouth once daily for 12 weeks and were followed up for another 12 weeks after cessation for treatment. At start and after 6, 12 and 24 weeks, patients were examined by upper endoscopy and carcinoids were counted and their size measured, and biopsies were taken from the oxyntic mucosa. Carcinoids were biopsied before study start and at treatment stop for histological examination and gene expression analyses. Blood samples were collected at treatment start and after 3, 6, 9, 12 and 24 weeks for measuring gastrin and chromogranin A as well as for safety assessment. Results: All patients had a reduction in the number and size of gastric carcinoids after 6 to 12 weeks treatment. There was a reduction in chromogranin A after 3 weeks treatment and a normalisation of chromogranin A after 6 weeks that was sustained during the remaining treatment period. Twelve weeks after cessation of YF476, chromogranin A was elevated to pretreatment levels. Blood gastrin levels did not change significantly during the study. There were no important adverse events. Results from histological examination and gene expression analyses of the oxyntic mucosa will be presented. Conclusions: Gastric carcinoids type 1 are caused by hypergastrinemia and targeted treatment with the orally active gastrin receptor antagonist YF476 seems promising. All patients in this phase 2 study had a reduction in carcinoid number and size after 12 weeks treatment and YF476 was well tolerated. Long-term administration of YF476 may be an effective and safe treatment alternative for this group of patients.

TFF1 Silencing Leads to Activation of B-Catenin/Tcf Signaling in Gastric Cancer Mohammed Soutto, Ahmed Katsha, DunFa Peng, M. Blanca Piazuelo, Pelayo Correa, Alexander Zaika, Wael El-Rifai Background: Trefoil factor 1 protein (TFF1) is a small secreted protein. It is constitutively and strongly expressed in the stomach, where it has a key role in maintaining mucosal integrity. Our previous studies using TFF1-knockout mice indicated a mucosal pro-inflammatory phenotype with a sequence of morphological and signaling changes leading to the development of gastric cancer. Methods and Results: In the current study, we hypothesized that the tumorigenic phenotype obtained in the TFF1-deficient gastric tissues is induced through regulation of b-catenin/TCF pathway. Using immunohistochemistry for b-catenin expression in the pyloric antrum region of the stomach in TFF1 wild type and TFF1 knockout mice, our results showed strong nuclear accumulation of b-catenin in the epithelial cells of TFF1 knockout mice; this accumulation was absent in TFF1 wild type. Using In Vitro cell models, we investigated the transcriptional regulation of b-catenin/TCF activity by TFF1 using the pTOP flash and its mutant pFOP flash luciferase reporter assays. MKN28 and SNU1 TFF1 stable cell lines showed 80% and 50% decrease in b-catenin activity, respectively, as compared to their corresponding pcDNA control cells (P<0.001). These results were confirmed using TFF1 adenovirus system in MKN28 cell line where transient TFF1 expression decreased b-catenin/TCF luciferase activity by 60% relative to control (P<0.01). Using quantitative real time PCR, we found that stable TFF1 expression led to a significant downregulation of b-catenin/TCF target genes, c-Myc and Cyclin D1 in MKN28 and SNU1 cell lines, as compared to their pcDNA control cells (P<.01). The immunofluorescence assay data indicated that reconstitution of TFF1 expression in MKN28 cell line by TFF1 adenovirus (5MOI) abrogated translocation of b-catenin to the nucleus and induced strong staining in the cell membrane as compared to the control cells where the b-catenin showed a strong staining in the nucleus. Conclusion: Our data suggest that b-catenin activation could play a role in the development of gastric neoplasms in TFF1 knockout mice. In addition, our In Vitro and In Vivo studies unveil a novel mechanism by which TFF1 regulates b-catenin/TCF activity, thereby implicating its tumor suppressor function in gastric cancer. Ongoing studies using In Vivo, ex-vivo and In Vitro models are underway to dissect the regulatory mechanisms that mediate TFF1 suppression of b-catenin/TCF.

58 Biomarkers of Human Stomach Metaplasia Progression Revealed by Proteomic Profiling of Paraffin-Embedded Tissues Josane F. Sousa, Amy-Joan L. Ham, Corbin W. Whitwell, Ki Taek Nam, Hyuk-Joon Lee, Han-Kwang Yang, Woo Ho Kim, Bing Zhang, Ming Li, Bonnie LaFleur, Daniel C. Liebler, James R. Goldenring The major proximate cause of gastric cancer is chronic Helicobacter pylori infection, which leads to a chronic inflammatory response and subsequent oxyntic atrophy (loss of acidsecreting parietal cells). The parietal cell loss leads to the development of two types of metaplasia: intestinal metaplasia (IM), characterized by the presence of cells with intestinal and goblet cell morphologies, and spasmolytic polypeptide-expressing metaplasia (SPEM) that shows morphological characteristics of the deep antral glands and expresses trefoil factor 2 (spasmolytic polypeptide). Both types of metaplasia in the stomach are associated with intestinal-type gastric cancer and are considered neoplastic precursors. However, the mechanisms driving the progression from metaplasia to neoplasia and even the relationship between the two metaplasia types remain unclear; although recent studies have suggested that IM arises in the context of pre-existing SPEM. While previous studies have focused on frozen tissue samples, marked improvements in technology have allowed us to examine the protein expression profiles of stomach metaplastic and neoplastic lesions from formaldehydefixed paraffin embedded (FFPE) samples. We performed a LC-MS/MS analysis after peptide isoelectric focusing (IEF) fractionation on a Thermo LTQ-Orbitrap to generate shotgun proteomic profiles from macrodissected FFPE samples of intestinal-type gastric cancer, metaplasia and normal mucosa (10 samples in each group). Based on a JT trend test (p < 0.01) we detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. In accordance with the parietal cell loss observed during metaplasia and cancer development, there was an overrepresentation of oxidoreductase activity and ion transmembrane transport functions among the proteins with decreased expression. The top 20 up-regulated proteins were preferentially represented by nucleic acid binding factors (NASP, PCNA, DDX5, NONO, RUBVBL2, RCC1) and cell surface or extracellular proteins (TNXB, PLEC1, FBLN5, LTF, DMBT1, F2, APOA1, ESYT1). Interestingly, we identified lactotransferrin (LTF) as a novel specific marker for SPEM and DMBT1 as a marker for intestinal metaplasia. In a tissue microarray containing 450 human gastric cancer samples, we observed that 71.7 % were positive for DMBT1, whereas 44.1% were positive for LTF. Both proteins showed significantly higher expression levels in stage I disease. In addition, the inverse relationship of LTF and DMBT1 expression in metaplastic glands containing both SPEM and intestinal metaplasia supports the concept that intestinal metaplasia develops from SPEM. This relationship suggests that metaplastic conversion is integral to the process of carcinogenesis in the stomach.

56 Zinc Finger Protein 545 is a Functional Tumor Suppressor Through Inhibiting Ribosomal RNA Transcription in Gastric Cancer Shiyan Wang, Wan Du, Xiaoxing Li, Qian Tao, Joseph J. Sung, Jun Yu Background & aims: Zinc finger protein 545 (ZNF545) is a member of Krüppel-associated box containing Zinc-finger proteins. Through a genome wide-screening, we identified a novel KRAB-ZFP member, zinc finger protein 545 (ZNF545), methylated in gastric cancer. However, the effect of ZNF545 in tumorigenicity was unexplored. In this study, we evaluated the expression profile, epigenetic regulation, biological function, molecular basis and clinic application of ZNF545 in gastric cancer. Methods: Promoter methylation of ZNF545 was evaluated by bisulfite genomic sequencing. The biological function of ZNF545 was determined by cell growth and apoptosis assays. ZNF545 target signal pathway was identified by promoter luciferase assay, Northern blot, run-on transcription assay, chromatin immunoprecipitation and co-immunoprecipitation assays. Clinical implication of ZNF545 was assessed in primary gastric cancers. Log-rank test and Cox regression model were used to identify independent predictors of prognosis. Results: ZNF545 was silenced or reduced in 16 out of 18 gastric cancer cell lines, but readily expressed in normal gastric tissues. Downregulation of ZNF545 was revealed to be mediated by promoter hypermethylation. Restoring ZNF545 expression in gastric cancer cell lines (AGS, MGC803) suppressed cell proliferation (P<0.01 in both cell lines) and induced apoptosis (P<0.01 in both cell lines) as compared with vector transfected cells. These effects by ZNF545 were attributed to inhibition of ribosomal RNA (rRNA) transcription. Inhibiting rRNA transcription by ZNF545 was further revealed to be associated with its direct ribosomal DNA (rDNA) promoter binding, recruitment of corepressor heterochromatin protein 1β and the reduction of trimethylated histone H3 at the lysine 4 residue at the rDNA locus. Moreover, ZNF545 methylation was detected in 53.2% (42/79) of gastric cancer tissues, 27.0% (20/74) of adjacent nontumor gastric tissues (P = 0.001), but none in 20 normal controls. Multivariate analysis revealed that patients with ZNF545 methylation in adjacent non-tumor areas had a significant decrease in overall survival. Kaplan-Meier survival curves showed that ZNF545 methylation was significantly associated with shortened survival in Stage I-II gastric cancer patients. Conclusions: we have identified a novel functional tumor suppressor gene ZNF545 inactivated by promoter methylation in gastric cancer. ZNF545 suppressed gastric cancer through silencing rRNA transcription by directly binding to rDNA promoter and recruiting co-repressor HP1β. ZNF545 methylation was associated with shorter survival in early stage of gastric cancers and may serve as a useful biomarker for patient with early stage gastric cancer.

59 Microbial Diversity of Gastrointestinal Flora Influences Dynamics of Gastric Cancer Progression in INS/GAS Mice Kvin Lertpiriyapong, Jennifer L. Lofgren, Sureshkumar Muthupalani, Yan Feng, Zhongming Ge, Mark T. Whary, Timothy C. Wang, James G. Fox A high percentage (~80%) of Helicobacter pylori (Hp) infected male INS-GAS mice raised in conventional housing developed gastric adenocarcinoma at 7 months postinfection (Pi). Germfree (GF) INS-GAS mice did not develop significant gastric lesions until 9 months old and did not develop GIN through 13 months. Hp monoassociation caused progressive gastritis, hyperplasia, dysplasia, and tissue proinflammatory immune responses between 5 and 11 months Pi. Eight of 18 male Hp monoinfected INS-GAS mice developed low to high-grade GIN by 11 months Pi. (Lofgren et al, 2011). We hypothesized that changes in gastric microbiota composition might promote GIN in achlorhydric stomachs of SPF mice. Three groups of INS/GAS mice were infected with Hp SS1 and control mice were sham dosed and followed for 7 months Pi: (1) GF mice (5M, 6F) (2). GF mice associated with 3 altered Schaedler flora-Clostridia spp., Bacteroides sp., and Lactobacillus sp. (9M, 7F) and (3) SPF mice (15M, 9F). All Hp infected groups had significantly (p<0.05) higher median gastric histology activity index (GHAI) scores than respective controls. SPF infected mice had significantly higher GHAI scores than ASF and GF groups of Hp infected mice. Importantly, none of the GF monoassociated Hp mice developed any high grade carcinoma when

57 Treatment of Gastric Carcinoids Type 1 With the Gastrin Receptor Antagonist YF476 Results in Regression of Tumours and Normalisation of Serum Chromogranin A Reidar Fossmark, Øystein Sørdal, Constantin S. Jianu, Gunnar Qvigstad, Malcolm J. Boyce, Helge Waldum Background and aims: Patients with chronic atrophic gastritis have long-term gastric hypoacidity and secondary hypergastrinemia. A proportion of them develop ECL-cell carcinoids in the oxyntic mucosa, which are classified as type 1 gastric carcinoids. Although many carcinoids remain indolent, some become more aggressive with time and metastasize. It is generally agreed that patients with type 1 carcinoids should undergo surveillance and some should be treated. The various treatment alternatives include use of somatostatin analogues and various surgical interventions, such as antrectomy. But less is known about which

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AGA Abstracts

AGA Abstracts

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