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INTEGRATED GENE EXPRESSION PROFILING AND IN SILICO EPIGENOMIC SCANNING TO ID~ENTIFY GENES INVOLVED IN MICROSATELLITE INSTABILITY STATUS OF COLORECTAL CARCINOMA Elena Deacu, Yuriko Mori, Fumiaki Sato, Florin M. Selaru, Andreea Olaru, ling "fin, Suna Wang, "/an Xu, Thomas C. IAu, David shihata, John M. Abraham, Stephen J. Meltzer

Background: Microsatellite instability (MSI) status is currently defined as either microsatellite- stable (MSS), low-frequency MSI (MSI-L), or high-frequency MSI (MS1-H). Hypermethylation of CpG islands in the promoter regions of several genes has been associated with MSI status in colorectal cancers. In order to identify novel methylation sites unique to MSI-H or MS1- L tumors in an unbused fashion, we conducted an expression profiling-based methylation target search. Methods: 41 primary colorectal cancers (12 MSI-H, 15 MSI-S and 14 MSI-L) were studied with high-density eDNA microarrays, and the bioinformatics methods principal components analysis (PC.A) and significance analysis of microarrays (SAM) were used to identify differentially expressed genes, which were in turn scanned for CpG islands in their putative promoter regions. Results: SAM and PCA identified 166 genes showing significantly different expression levels in MSI-H vs. non-H or in MSI-L vs. MSS colon tumors. 95 of these genes were decreased in mRNA expression in either MSI-H or MSI-L tumors. Genomic chtahase screening revealed that of these 95 genes, 48 had CpG islands within their putative promoter regions. These 48 genes were scanned for expression in normal tissue and for likely cancer-related putative protein functions. This combined approach identified 19 genes vath both CpG islands and significantly differential expression levels between MSI categories. 10 genes were excluded because of pro-carcinogenic (oncogenic) functions. Among the remaining 9 genes, 2 were underexpressed in MSI-L vs. MSS tumors: RBM8A (a binding partner of potential tumor suppressor gene OVCA1) and OAS3 (an IFN-induced protein required to degrade RNA). 7 were underexpressed in MSI-H vs. non-H tumors: RAB32 (ras family gene), PTPRO (a receptor-protein tyrosine phnsphatase), SDBCAG84 (breast cancer anugen 84), HDACll (histone deacetylase), ITPR2 (inositol triphosphate receptor), N33 (putative prostate cancer tumor suppressor), and TSC22 (TGFI~ stimulated transcription regulating factor). Conclusions: The anti-carcinogenic functions of these genes suggest that they are potential targets of tumor-specific methylation. In support of this hypothesis, one gene (N33) is a known tumor suppressor reactivated by promoter hypermethylation in multiple cancers. These results suggest that our approach will identify novel genes involved in the MSI status of colorectal carcinoma.

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The Role of KmppeI-Like Factor 6 (KLF6) in Colorectal Neoplasia Helen Reeves, Michael Arthur, Goutham Narla, Olagunju Oginbiyi, Asif Haque, Amanda Katz, Shlomitt Goldberg, Noam Harpaz, John Martignetti, Scott Friedman

We have recently described the ubiquitous transcription factor, KLF6, as a novel tumor suppressor gene inactivated in the majority of prostate cancers (Narla Get a], Science 294, 2563-2566, 2001). In the present study we report that KLF6 is inactivated in over 50% (26/50) of both sporadic and inflammatory bowel disease (IBD) related colorectal cancers (CRC) by a combination of loss of haterozygosity (LOH; 82%) and mutation of the second allele (56%). Furthermore LOH was present in non-tumour IBD-affected colonic epithelium (7/8 cases) in patients with CRC and in 65% of colonic adenomas (17/26 < lcm, varying degrees of dysplasia) suggesting that this occurs as an early event in the pathogenesis of CRC KI.F6 is present at low levels in normal colonic epithelium, but abnormal cytoplasmic accumulation was detected in all 50 CRC cases. Cellular transfection of CRC-derived KLF6 tamour mutants led to cytoplasmic rather than nuclear accumulation, suggesting that intracel- lular mislocalization is directly related to mutations of KLF6 We have further explored the mechamsms by which KLF6 inactivation may contribute to carcinogenesis. Wild-type KLF6 slgnificandy suppressed cell growth in both colonic fibroblasts (30%, p<0.O01 ) and colorectal cancer cell lines (HT29, HCT116). This was associated with a 2-3 fold increased expression of the cell cycle inhibitor p2i/WAF and down-regulation of the proliferative epidermal growth factor receptor (EGF-R), thereby favouring growth inhibition, tn addition, matrix metalloproteinase-2 (MMP-2) mRNA and protein was decreased, with a corresponding 50- 60% decrease in MMP-2 activity. In contrast, the CRC-derived mutant isoforms of KLF6 were pro-proliferative, failed to upregulate p21/WAF, and increased rather than suppressed the levels of the EGF-R and MMP-2. These CRC-derived KLF6 mutant mediated effects therefore increase both the proliferative and invasive capacity of tumour cells. Collectively these data indicate that KLF6 .inactivation plays a key role in the pathohiology of CRC.

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Mice with a Defective SHP2 Binding Site on the IL-6 Co-Receptor gp130 Develop Gastric Adenomas with Dysplasia and Submucosal Invasion LouLse M. Judd, Barbara Alderman, Meegan Howlett, Jifiian Moverley, Brendan Jenkins, Matthias Ernst, Andrew S. Giraud

IL-6 family cytokines exert their pleiotrophic effects through activation of multiple subunit receptors. The common gp130 subunit signals via discrete modules resulting in activation of STAT 1/3, or ras/erk after SHP2 binding. A knock-in mutation of residue p-Y757 on gpl30 prevents SHP2 binding thus inactivating ragerk signaling and resulting in reciprocal up-regulation of STAT3 expression (FF mouse). Mutants developed normally until weaning, gastric histology at 4-6 weeks revealed antra] hyperplasia consistent with adenoma formation, the fundic stomach appeared normal with typical numbers of parietal and chief cells (H/K- ATPase and IF IHC). Circumferential lesions were composed of elongated foveofae and branching glands with progressive dyspiasia. Adenomas increased in size from 13+3 to 198+22 mm ~ between 6 and 16 weeks and then growth slowed. By 38 weeks fundic involvement was apparent with depleted parietal and chief cells, adenomas encompassed the entire glandular stomach, occluding the pylorns and reducing the ability to thrive. High grade dysplasia, including nuclear stratification, and submucosal glands were observed, but metastasis to lymph nodes were not apparent. Cellular proliferation was markedly increased (PCNA) with no increase in apoptosis (TUNED. Gastrin mRNA, serum gastrin and G cell

numbers were reduced, initially the pH of the gastric contents was comparable to wild type mice but it rose sharply at 18 weeks. The expression of the tumor suppressor TFF1 in antra] extracts of FF mice was reduced 70% at 12 weeks (RIA), mRNA quantification (ISH and Northern) showed a similar reduction, suggesting transcriptional regulation by IL-6 family cytokines. In contrast, the growth factor Reg I mRNA was massively upregulatedin developing adenomas. Unlike FF mutants, mice heterozygous for the mutation developed smaller gastric adenomas predominantly in the fundic mucosa, with lower penetrance (5/25) and delayed development (> 20 weeks). Conclusions: 1. The dominant phenotypic consequence of gp130-ras/erk inactivation is antral stomach adenoma that develops with little fundic involve- ment until the advanced stages. 2. Inactivation of the SHP2 binding site on gp130 leads to loss of ras/erldAP-1 signaling and reciprocal STAT3 activation, resuhmg in reduced pS2/ TFF1 gene expression. 3. Paradoxically, gastrin gene expression is reduced and Peg I gene expression enhanced in FF mice, suggesting indirect regulation by IL-6 family cytokines.

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Frameshift Mutations in Gastric Epithelial Cells Are Caused by Mismatch Repair Deficiency Induced by Helicobaeter Pylori Infection Antonia R. Sepulveda, Hong Tao, Dong Park, Yao Yuan, Benjamin 8urkhead, Jeorge Seputveda

Background: H pylori (Hp) infection causes chronic gastritis and is a major risk factor in the development of gastric cancer Deficiencies of mismatch repair (MMR) are associated with frameshift mutatio~ that can be detected as microsatelhie instability (MSI). Active Hp infection is present more frequently in individuals with MSI-posinve gastric cancers, raising the possibility that Hp infection causes MSI-type mutations in the gastric epithelium. We found that Hp infection of cultured gastric cancer cell lines causes a marked drop in the levels of MutL and MutS DNA MMR proteins to less than 10% of normal levels. Aims: The aim of this study was to determine whether the drop in MMR protein levels induced by H. pylori is associated with MSl-type frameshift mutation accumulation. Methods: Mutations were detected using a reporter vector (pcDNA3. I-(CA)13-EGFP), in which a (CA)13 repeat interrupts the EGFP reading frame. Frameshift mutations in the (CA)13 repeat restore the EGFP reading frame and result in EGFP-posinve cells. The gastric cancer cell line AGS was stably transfected with the EGFP reporter carrying a CA(13) repeat in the coding region. AGS cells were co-cultured with Hp (ATCC 43504) for 48 hours, recovered, passed, and this cycle of infection was repeated up to 5 times. The number of EGFP-posinve cells in Hp infected and non-infected control were determined by FACS analysis. The amounts of EGFP protein in Hp infected and non-infected controls were determined by western blot analysis. Expression of the two main DNA mismatch repair proteins hMLH1 and hMSH2 was determined by western blot. Results: The levels of EGFP protein significantly increased in AGS ceils infected by H. pylori, and the numbers of EGFP-positive ceils were significantly higher in AGS cells infected by H pylori as compared to controls. Sequence analysis of isolated EGFP-positive cell clones showed a frameshift in the CA13 repeat with loss of repeat elements, confirming that EGFP expression was associated with a restored EGFP reading frame. Conclusion: This is the first report showing that infection of gastric epithelial cells by H. pylori induces frameshift mutagenesis. This effect results from impaired DNA MMR caused by a major drop in MMR protein levels induced by H. pylori organisms and is likely to be an important molecular mechanism underlying gastric carcinogenesis related to H. pylori gastritis.

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Oncogenic RAS Induces a Senescence-Like Phenotype in Human Esophageal Epithelial Cells Munenori Takaoka, Hideki Harada, Ben Rhoades, Therese B. Deramaudt, Oliver G. Opitz, Gregory H. Enders, Hiroshi Nakagawa

Introduction: Activation of oncogenic ras induces a senescence-like phenotype in normal fibroblasts and epitheha] cells independent of telomere length. Such an oncogene mediated activation of senescence may represent a tumor suppressive mechanism. In mouse epidermal keratinocytes, the ARF-p53 pathway is required in the induction of ras mediated activation of senescence. However, it remains unclear how ms oncogenic activation affects human esophageal ceils (keratinocytes) where p53 mutation is common in esophageal squamous cell carcinoma (ESCC). Methods: Primary human esophageal cells (EPC2) or mouse esopha- geal cells (MEC) isolated from wild-type or p5Y / mice were cuhured in serum free medium, and transduced with retrovimses expressing tdomerase (hTERT), dominant negative p53 (p53atT~u), or oncogeinc ras (H-rasVl~). Senescence was confirmed by [3-galactosidase (SA- [3gal) staining. Cell proliferation was assessed by 3H-thymidine incorporation assay. Protein expression was determined by Western blotting. Results: EPC2 cells expressing hTERT (EPC2-hTERT) were immortalized and had evidence of intact retinoblastoma (pRb) and p53 functions. In EPC2-hTERT cells further transduced with p53 RIT~H, hydroxyurea or ultraviolet treatment failed to induce p21 wA~t and Bax, confirming the dominant negative effect of p53 R~75~. Ras transduced EPC2 or EPC2-hTERT cells showed senescence-like morphology (100% cells) and decreased cell proliferation within 72 hours, regardless of p53 status. They remained quiescent and were positive for SA-[3gal staining (30-40% cells) within two weeks. Oncogeinc ras induced hypophosphorylation ofpRb in EPC2-hTERT expressing either wild- type or mutant p53. p16 ~NK4A was also induced in EPC2-hTERT expressing wild-type p53, suggesting that p16 ~n~*A is essential in ras mediated induction of the senescence-like pheno- type. Interestingly, p14 ̂ aF was downregulated in ras transduced EPC2-hTERT cells. Wild- type MEC also underwent ras-mediated activation of senescence. By contrast, more than 80% ofras transduced p53 ~ MEC continued to prohferate. Conclusions: In human esophageal cells or keratinocytes, oncogenic ras induced senescence through activation of p16, but appeared not to require the ARF-p53 pathway. Therefore, ras mediated senescence involves distinct mechanisms between human and mouse esophageal keratinocytes. Inactivation of the pRb pathway may be necessary for ras to transform esophageal epithelial ceils and overcoming senescence.

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