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sSa1682

TNF-α-Mediated Down-Regulation of CDx2 Decreases the Expression of β-Catenin Degradation Complex Genes in Colorectal CancerMehmet Coskun, Anders K. Olsen, Michael Bzorek, Susanne Holck, Ulla H. Engel, OleH. Nielsen, Jesper Troelsen

Background: Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that is highlyup-regulated in inflammatory bowel disease and reduces the expression of the intestinalspecific homeodomain transcription factor, CDX2. CDX2 is a master regulator of the intestinalhomeostasis and is considered to be a tumor suppressor. WNT/ β-catenin signaling is criticalfor intestinal cell proliferation, but decreased CDX2 expression contributes to an abnormalincreased activation of WNT signaling and progression of cancer. Furthermore, low CDX2expression is associated with enhanced epithelial-to-mesenchymal transition (EMT) in colo-rectal cancer (CRC). Inflammation is often observed at the invasive front of the colon cancer,and it has been suggested that the inflammatory microenvironment around a tumor alsoinduces EMT. Hence, it is likely that down-regulation of CDX2 expression in the invasivefront in CRC contributes to enhanced tumorigenicity by regulating genes associated withthe WNT/β-catenin pathway. Aim: To determine the role of TNF- α in the regulation ofCDX2 expression in CRC and its influence onWNT/β-catenin signaling. Methods: Carcinomaspecimens were obtained from patients undergoing surgical resection of the rectum. Tencases with tumor cell buddings were selected, and the expression patterns of TNF- α andCDX2 at the invasive front were evaluated on immunostained slides. The mechanisms behindTNF-α-mediated down-regulation of CDX2 were investigated in vitro by selective inhibitors,and the impact of TNF-α on APC, AXIN2 and GSK3β expression were analyzed by quantita-tive real-time PCR (qPCR) in Caco-2 cells. Subsequently, in vivo CDX2-DNA interactionsto APC, AXIN2 and GSK3β gene regulatory elements were investigated by chromatin immu-noprecipitation in relation to the TNF-α status (presence vs. absence). Results: Immunohisto-chemical staining showed reduced CDX2 positive-cells in tumor buddings in areas withTNF-α expression in the surrounding inflammatory cells. TNF-α treatment showed a dose-dependent decrease of CDX2 mRNA and protein expression dependent on p38 and NF-κB, but not on JNK and ERK signaling pathways. Down-regulation of CDX2 leads tosignificantly decreased levels of APC (p,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05)mRNA. In line with these results, TNF-α treatment impaired the ability of CDX2 to interactand activate the expression of APC (p ,0.05), AXIN2 (p,0.01), and GSK3β (p,0.05).Conclusions: This study suggests that TNF-α has a tumor-promoting effect on CRC byinfluencing the activity of WNT signaling through down-regulation of CDX2 expression.These findings provide a novel insight into the molecular regulation of genes in the β-catenindegradation complex in response to the pro-inflammatory cytokine TNF- α in cancer cells.

Sa1683

Five Different Subclasses of Colorectal Neoplasias Identified by IntegratedGenetic and Epigenetic Analysis: Association With Polypoid and NonpolypoidCarcinogenesisKenichi Konda, Kazuo Konishi, Atsushi Katagiri, Masayuki Tojo, Yutaro Kubota, TakashiMuramoto, Yuichiro Yano, Yoshiya Kobayashi, Toshihiro Kihara, Kensuke Shinmura,Teppei Tagawa, Toshiko Yamochi, Yoichi Ito, Michio Imawari, Hitoshi Yoshida

Background & Aims: Colorectal adenoma develops to cancer with the accumulation ofgenetic and epigenetic abnormalities. In addition, colorectal adenomas can be morphologi-cally classified into polypoid neoplasias (PNs) and nonpolypoid neoplasias. They showdifferent clinicopathological and molecular features. The aim of this study is to investigatewhether the epigenetic and genetic features of colorectal cancer (CRC) are shared withseveral subtypes of conventional neoplasias (adenomas and Dukes' A carcinoma). Methods:We evaluated both genetic alterations (mutations of KRAS, BRAF, and p53; and microsatelliteinstability [MSI]) and epigenetic (methylation status of 9 genes/sequences, including theCpG island methylator phenotype [CIMP] marker) in 158 CRNs (81 low-grade dysplasiasand 77 high-grade dysplasias or Dukes' A cancers). A clustering analysis on the basis of acombination of genetic and epigenetic profiling was performed to identify subclasses withmolecular signature. Results: Unsupervised hierarchical clustering analysis of genetic andepigenetic data identified 5 distinct groups of CRNs. Cluster 1 cases showed a higherfrequency of BRAF mutation (50%), CIMP (50%), and high-frequency MSI (83%) but noKRAS or p53 mutations, whereas cluster 3 cases revealed a higher frequency of p53 mutation(100%), lower methylation level of LINE-1, few KRAS mutations (14%), and an absence ofCIMP and high frequency MSI. KRAS mutations were frequently observed in cluster 4 cases(97%), whereas cluster 5 cases showed higher frequencies of KRAS mutation (64%) andCIMP (100%) and a higher methylation level of MGMT. However, we observed few geneticand epigenetic alterations in cluster 2 cases. Regarding associations with clinicopathologicalfindings, cluster 1 and 3 cases showed higher frequencies of nonpolypoid neoplasias, whereascluster 4 and 5 cases more frequently displayed polypoid neoplasias. Conclusions: Wedemonstrated that CRNs were classified into 5 distinct molecular subclasses and foundclinicopathological differences among these subclasses, suggesting that different mechanismsare involved in colorectal tumorigenesis.

Sa1684

Common Food Additive Carrageenan Stimulates Wnt/β-Catenin SignalingPathway in Colonic Epithelium by Inhibition of Nucleoredoxin ReductionJoanne K. Tobacman, Sumit Bhattacharyya

Wnt signaling is associated with increased cell proliferation in development and in malig-nancy. Previously, exposure to the common food additive carrageenan was associated withincreased Wnt9a expression and increased cytoplasmic β-catenin in human colonic epithelialcells, and carrageenan exposure has been associated with colonic neoplasms in animal modelsin numerous reports. In this study, exposure of the NCM460 human colonic epithelial cellline and of C57BL/6J mice to low concentrations of carrageenan stimulated the Wnt/ β-catenin signaling pathway, leading to significant increases in nuclear β-catenin, activated

S-282AGA Abstracts

TCF/LEF, and cyclin D1 expression. These effects were mediated through carrageenan-induced reactive oxygen species (ROS) and inhibited by the reactive oxygen species scavengerTempol. This investigation of the mechanism by which carrageenan exposure and theassociated increase in ROS could stimulate the Wnt signaling pathway focused on nucleore-doxin (NRX; thioredoxin 4) and thioredoxin reductase activity. NRX impacts on the cyto-plasmic β-catenin degradation complex due to its interaction with dishevelled (DVL), whichpreferentially binds with reduced NRX, rather than with oxidized NRX. Unbound DVL caninteract with axin, and thereby lead to β-catenin nuclear translocation by effect on the axin-APC-GSK3β-casein kinase cytoplasmic complex. Reduced nucleoredoxin declined to ~40%of baseline (p,0.001) following carrageenan exposure. Inhibition of thioredoxin reductaseby either carrageenan (1 μg/ml x 24 h) or H2O2 (0.1 mM or 1.0 mM x 1 h) reduced co-immunoprecipitation of NRX with DVL, and disruption of the DVL-NRX complex. The ratioof free (oxidized S-S) NRX to bound (reduced SH) NRX increased from 0.4 to 2.4 followingcarrageenan, consistent with release of DVL from NRX, and association of DVL with thecytoplasmic β-catenin destruction complex and disinhibition of the nuclear translocation ofβ-catenin. When NRX was silenced by siRNA, nuclear β-catenin increased (p,0.001). BothNRX silencing or carrageenan exposure significantly inhibited thioredoxin reductase activity(p,0.001), and the combination of NRX silencing and carrageenan had no increased inhibi-tory effect. In addition to effects on the Wnt/ β-catenin signaling cascade, carrageenan expo-sure stimulated the mRNA expression of wnt9a in mouse colonic epithelium to 2.4 timesthe baseline (p,0.0001, unpaired t-test, two-tailed). In the NCM460 cells, carrageenanexposure increased Wnt9A expression to 2.8 ± 0.3 times the baseline (p ,0.001). Wnt9Aprotein increased to ~2.9 times the baseline in the carrageenan-treated NCM460 cells. Thus,carrageenan exposure both increased the expression of Wnt9A and activated Wnt signalingin colonic epithelium. These findings suggest how a common dietary ingredient can contributeto colon carcinogenesis.

Sa1685

Chromatin Remodeling via Sin3-HDAC and Hp1-Hmt Pathways in theRegulation of Oncogenic AKT1Anamay N. Sharma, Sonia Chowdhury, Gwen A. Lomberk, Cathrine J. DeMars, Prasad G.Iyer, Paul J. Limburg, Kausilia K. Krishnadath, Kenneth K. Wang, Raul A. Urrutia, NavtejButtar

BACKGROUND: We have recently shown that AKT1 phosphorylation at Thr-308 or Ser-473 by upstream oncogenic kinases as well as increased AKT1 expression play a criticalrole in injury-induced neoplasia in Barrett's esophagus. Even though there is plethora ofliterature on the regulation of AKT1 activation through its phosphorylation, the mechanismsof regulation of AKT1 expression remain an underrepresented research area. We found thattranscription factor KLF11 binds to and down regulates AKT1 expression to exert growthinhibition in Barrett's esophagus. The AIM of this investigation was to examine key chromatinremodeling mechanisms that regulate AKT1 expression. METHOD and RESULTS: Sincethere are no gene amplifications or rearrangements at AKT1 locus in patients with esophagealadenocarcinoma, We used chromatin immuno precipitation assay (ChIP) and found thatduring neoplastic changes in Barrett's, while AKT1 promoter DNA methylation status didn'tchange, native AKT1 promoter chromatin showed marked increase in histone 3 (H3K18and K27) acetylation H3K4 methylation (gene activation mark), as well as decrease in H3K9methylation (gene repression mark) . We also found that the restitution of growth inhibitorytranscription factor KLF11 in neoplastic Barrett's epithelial cell decreased H3 acetylationbut increased H3K9 methylation on AKT1 promoter. Since KLF11 is typically lost duringneoplastic progression, these findings suggest that a loss of KLF11 mediated regulation atthe level of chromatin remodeling could be involved in AKT1 overexpression. Congruentwith this, using promoter-reporter luciferase assay, we found that KLF11 mutant that doesnot recruit Sin3-HDAC to deacetylate AKT promoter failed to completely represses AKT1promoter activity. Similarly, KLF11 mutant that does not recruit HP1-HMT pathway toplace repressive H3K9 methylation marks did not effectively repress AKT1 promoter activity.Since Sin3-HDAC and HP1-HMT recruitment by KLF11 is modifiable through membraneto nuclear signals, our findings suggest that either through the loss of KLF11 or via alteredrecruitment of chromatin remodelers by KLF11 play an important role in dysregulation ofAKT1 expression during neoplasia in Barrett's esophagus. CONCLUSION: Our findingsprovide novel translational targets to correct AKT1 expression to prevent neoplasia inBarrett's esophagus.

Sa1686

Characteristics of and Associations Among the HER2 Signal-Related FactorsThought to Be Predictive Factors for the Effectiveness of TrastuzumabTherapy in Gastric CancerMiki Ito, Yasutaka Sukawa, Katsuhiko Nosho, Hisayoshi Igarashi, Takafumi Naito, HiroakiKunimoto, Kei Mitsuhashi, Mayumi Nakazawa, Wakana Sumioka, Yasushi Adachi,Masashi Mikami, Mariko Kawayama, Hiromu Suzuki, Hiroyuki Yamamoto, YasuhisaShinomura

Background: An anti-HER2 antibody, Trastuzumab (Tmab), was proved to be effectiveagainst gastric cancer (GC) with HER2 overexpression. Based on research in breast cancer,HER2-related factors including expression of HER2 truncated extracellular domain(p95HER2), alterations in the PI3K-Akt pathway such as PIK3CA mutation or PTEN inactiva-tion, and cross talk between HER2 and other receptors such as IGF1R have been receivingincreasing attention as predictive factors for the effectiveness of Tmab therapy in GC.Aims: We aimed to systematically characterize HER2-related factors and to determine theirprevalences and relationships with clinicopathological andmolecular features, such asmicros-atellite instability (MSI) and pAkt expression. Methods: We analyzed 231 T2-T4 GC tissues.Expressions of HER2, PTEN, IGF1R and pAkt were analyzed by immunohistochemistry(IHC). PIK3CA mutations in exons 1, 9 and 20 were analyzed by pyrosequencing. p95HER2expression was analyzed in HER2-positive cases (IHC 2+ and 3+) by immunofluorescencestaining with an anti-HER2 antibody directed against the HER2 internal domain. Results:HER2 overexpression (IHC 3+) was present in 20 samples (8.7%) and was significantlycorrelated with intestinal histological type (p=0.05). PIK3CA mutations were present in 20