(from 100% to 41.7%, P<0.05). The downstream targets of ZNF331 were identified by 2-DE in stably transfected MKN28 cells. Among the 33 differential protein spots, nine werefunctional importance and further validated by real-time RT-PCR and/or western blot. Thetumor suppressive effect of ZNF331 is mediated by down-regulation of genes involved incell growth (EIF5A, GARS, DDX5, STAM, UMPS) and migration/invasion (ACTR3, DSTN),and up-regulation of genome stability gene (SSBP1) and cellular senescence gene (PNPT1).Conclusion: These results suggest that ZNF331 possesses important functions for the suppres-sion of gastric cancer and can act as a functional tumor suppressor gene in gastric carcinogen-esis. Acknowledgment The project was supported by Research Grant ERG CUHK (GRF473008).

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Integrated Genome-Wide Analysis of Promoter Methylation and FOXD3Binding in Gastric CancerAlfred S. Cheng, May S. Li, Wei Kang, Jian-Liang Chou, Yuen Yee Cheng, Tim H. Huang,Jun Yu, Ka Fai To, Joseph J. Sung, Michael W. Chan, Francis K. L. Chan

Background: Gastric cancer is among the most lethal malignancies worldwide. Epigeneticsilencing of tumor-suppressors by promoter hypermethylation has emerged as an importantmechanism in gastric carcinogenesis. Forkhead box (Fox) proteins are a superfamily ofevolutionarily conserved transcriptional regulators that control essential developmental andhomeostatic processes. Consequently, a loss or gain of Fox function can lead to carcinogenesis.Major findings: Using a genome-wide approach called Microarray-based Methylation Assess-ment of Single Samples (MMASS), Fox factor FOXD3 has been found to be a novel hyperme-thylated gene in primary gastric cancer. Quantitative methylation-specific PCR confirmed asignificant elevation of FOXD3 methylation levels in gastric cancer compared to paired non-tumor tissues (65 pairs; p < 0.0001). The average levels of FOXD3 methylation in 65 tumor-adjacent tissues and 28 precancerous intestinal metaplasia lesions were significantly higherthan that in 23 control H. pylori-negative gastric tissues (both p < 0.0001). The clinicalimportance of this early carcinogenic event was evidenced by the significant associationbetween elevated FOXD3 methylation in tumor-adjacent tissues and shortened survival ofgastric cancer patients (p = 0.02). Western blot and immunohistochemistry showed thatFOXD3 protein was strongly expressed in normal human stomach tissues but silenced ordown-regulated in 69% (78/113) of primary gastric cancers and all 11 examined gastriccancer cell lines. Treatment with 5'aza-2'-deoxycytidine reactivated FOXD3 expression insilenced cells, indicating that FOXD3 expression is controlled by methylation. Functionalassays demonstrated that FOXD3 significantly reduced the growth and invasiveness of AGSand MKN1 gastric cancer cells. Ectopic FOXD3 expression induced activation of caspase-3 and -8 accompanied by cleavage of PARP, while knockdown of FOXD3 by small interferingRNA decreased PARP cleavage. To explore the growth-inhibitory mechanisms by FOXD3,chromatin immunoprecipitation (ChIP) coupled with human promoter arrays was performedand 66 significant FOXD3 direct target genes were uncovered (p < 0.001), including aknown FOXD3 target ID4. Interestingly, gene ontology analysis revealed that FOXD3 targetgenes were predominantly involved in apoptosis regulation (p < 0.05). Quantitative ChIP-and RT-PCR further showed that the caspase-dependent apoptotic regulators CYFIP2,MAP2K6, and RARB were directly up-regulated by FOXD3 in gastric cancer cells and down-regulated in a subset of primary gastric cancers. Conclusions: Our integrated epigenomicanalyses revealed that promoter hypermethylation-mediated silencing of FOXD3 plays acrucial role in gastric carcinogenesis via deregulation of downstream caspase regulators andthe associated apoptotic pathway. This study was supported by RFCID (08070172).

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Quantitative Analysis of Hydroxymethylated DNA in Primary ColorectalCancerYotaro Kudo, Keisuke Tateishi, Keisuke Yamamoto, Shinzo Yamamoto, Dai Mohri,Yoshihiro Isomura, Yoshinari Asaoka, Hideaki Ijichi, Masao Omata, Kazuhiko Koike

DNA methylation is a covalent chemical modification adding a methyl group at the carbon5 position of cytosine ring in CpG dinucleotides. It plays important roles in transcriptionalregulation in mammalian cells, and the profiles of DNA methylation in genome is profoundlyaltered in neoplasia. In particular, there are a lot of intensive researches about the pivotalrole of aberrant promoter hyperrmethylation, which is associated with inappropriate genesilencing, in the characteristics of malignant cells, and overexpression of methylating enzymes,DNA methyltransferases (DNMTs) 1, 3A, and 3B is reported in human malignancies. Inaddition, the mutation of DNMT3A was finally found in malignant cells. In a while, theenzymes responsible for DNA demethylation have been elusive for a long time, and so theexistence of active DNA demethylation itself has been obscure so far. Recent studies demon-strate that human TET1 and TET2 proteins and mouse Tet proteins (Tet1, Tet2 and Tet3)are capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC),raising the possibility that TET proteins mediate DNA demethylation process in mammaliancells. However, the biological significance of 5-hmC for gene expression or biological pro-cesses is totally unknown. Interestingly, the mutation of TET2 is reported in myeloid cancers,suggesting a possibility that the molecular functions of TET family proteins are involved inoncogenesis or tumor progression. To address the significance of TET proteins in digestivecancers, we examined the expression of TET family genes (TET1, TET2 and TET3) andDNMT family genes (DNMT1, DNMT3A and DNMT3B) using quantitative PCR analysis intwenty-two surgically resected primary colorectal cancers and adjacent normal mucosa. Alsowe established an assay system estimating the global level of 5-hmC in clinical samplesusing dot blot technique. Mutational analysis of KRAS and BRAF was performed with directsequencing method. The clinicopathological parameters were applied to those moleculardata. Seventy-three percent (16/22) of colorectal cancers display decreased level of 5-hmC.Reduced TET1 expression is found in half of the colorectal cancers (11/22), which is tightlyassociated with reduced TET3 expression (P < 0.001). Cancers with reduced TET1 expressionare found mainly in proximal colon (60% (6/10)) (P < 0.05). KRAS mutant and BRAF mutanttumors are found in 31.8% (7/22) and 9.1% (2/22) cases, respectively, however, our datashow no correlation between TET1 expression level and these genes mutation status, neither

S-819 AGA Abstracts

nor age and sex of patients, Dukes' grade, or tumor grade. Our data suggest that the decreaseof TET proteins and 5-hmC level might play some roles in colorectal cancers.

Tu1721

Identification of Mirnas and Their Gene Targets Differentially Expressed inMicrosatellite Stable and Unstable Colorectal Cancers Through an IntegratedAnalysisRosa M Xicola, Wenbo Mu, Lei Huang, Vanessa R. Sohn, Brian J. Doyle, Rodrigo Jover,Angel Carracedo, Montserrat Andreu, Xavier Bessa, Antoni Castells, C. Richard Boland,Ajay Goel, Epicolon Investigators, Yang Dai, Xavier Llor

Background: The two main types of colorectal cancer (CRC) develop either through microsa-tellite instability (MSI) due to mismatch repair deficiency or through chromosomal instabilitywith tumors being microsatellite stable (MSS). These types have biologically distinct carcino-genic pathways, which result in different prognosis and therapeutic responses. Some miRNAshave been found to have an altered expression in CRC. miRNAs are non-coding RNAs thatfunction as posttranscriptional regulators and are implicated in carcinogenesis. Each miRNAcan potentially regulate the expression of different genes. Objective: To identify differentiallyexpressed miRNAs likely responsible for unique biological features of MSS and MSI tumorsthrough an integrated analysis of miRNA and gene expression. Methods: 26 sporadic CRC(20 MSS, 6 MSI), and 7 normal colon tissue were included. All samples were analyzed withthe Affymetrix GeneChip human genome ST 1.0 array and the Taqman miRNA human poolA array to profile gene and miRNA expression respectively. The raw Ct values of miRNAon each assay were normalized by endogenous controls and data for all 33 samples wasquintile normalized using HTqPCR package. mRNA data was processed and normalizedusing RMA package. To further identify the functional targets of a miRNA, we searched inthe set of the differentially expressed mRNAs whose expression profiles were reverselycorrelated with that of the miRNAs. Differentially expressed miRNAs and mRNAs wereidentified based on Limma package by a significant threshold 0.05 for adjusted p-values.TargetScanHuman (v5.1) was employed for the prediction of miRNA targets. Results: Weidentified 18 differentially expressed miRNAs that inversely correlated with the expressionof a set of target genes that differentiated normal from tumors. On the other hand, weidentified 22 miRNAs and 327 mRNAs differentially expressed between MSS and MSI CRC.5/22 miRNAs reversely correlated with a differential expression of target genes. 3/5 miRNAsand their biological targets differentiate the two types of tumors and the normal tissue. has-miR-299-3p RNF43 Conclusion: This study identified miRNAs that are not only uniquelyderegulated in the two types of CRC but they likely play a significant biological role as theirtarget genes result in a significantly differential expression also in comparison with normaltissue. Thus, the 3 genes can be implicated in their different carcinogenic processes as RBM7is a RNA binding protein, PMEPA1 has been implicated in Wnt signaling and RNF43 hasa growth promoting effect. Further experiments will identify possible tumoral biomarkersamong the 18 differentially expressed miRNAs and also will assess how these miRNAsregulate tumor development.

Tu1722

Epigenetic Inactivation of Calcium-Sensing Receptor in ColorectalCarcinogenesisYasutaka Sukawa, Hiroyuki Yamamoto, Katsuhiko Nosho, Chie Miyamoto, NobukiMiyamoto, Hiroaki Taniguchi, Yasushi Adachi, Mayumi Nakazawa, Hiromu Suzuki,Shigeru Sasaki, Yoshiaki Arimura, Yasuhisa Shinomura

We have previously reported the genetic and epigenetic alterations in colorectal cancer(Nature, Science, Nature Genetics 2006, 2009, Cancer Cell 2010). Ca2+ is a chemopreventiveagent for colrectal cancer. Ion transport systems are often altered in human cancer. The aimof this study was to clarify the alterations of calcium-sensing receptor (CASR), a memberof the G protein-coupled receptor family, in colorectal carcinogenesis. We analyzed theexpression of CASR in colorectal cancer cell lines and in cancer and adenoma tissues byRT-PCR and immunostaining. By using bisulfite sequence analysis and methylation-specificPCR, we analyzed methylation of the CASR promoter. CASR mRNA and protein expressionwas significantly downregulated in most of the cancer cell lines. CpG islands were denselymethylated in cancer cell lines with reduced CASR mRNA expression. Treatment with ademethylating agent, 5-aza-2'-deoxycytidine, and/or a histone deacetylase inhibitor, trichosta-tin A, restored CASR expression in the cancer cell lines. Disruption of CASR expression inCASR-unmethylated HCT-8 cells blocked the enhancing effect of Ca2+ on the cytotoxicresponse to 5-fluorouracil. CASR expression was observed in normal colonic epithelialcells and was retained in most adenoma tissues. CASR mRNA and protein expression wassignificantly downregulated in cancer tissues. There was an inverse relationship betweenCASR expression and degree of differentiation. Immunohistochemical CASR staining wasreduced more predominantly in less-differentiated cancer tissues and/or in cancer cells atthe invasive front, where nuclear/cytoplasmic β-catenin was often localized. CASR methyl-ation was detected in 69% of colorectal cancer tissues and 90% of lymph node metastatictissues and was significantly correlated with reduced CASR expression. CASR methylationwas also detected in 32% of advanced adenoma tissues but was detected in only 9% ofadenoma tissues and was not detected in hyperplastic polyp tissues. CASR methylationappears to occur at an early stage and progress in colorectal carcinogenesis. The results suggestthat epigenetic inactivation of CASR plays an important role in colorectal carcinogenesis.

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