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s952

Nardilysin Maintains Gastric Cancer Cell Growth via Promoting Shedding ofTNF-α and Intrinsic Cytokine SignalingKeitaro Kanda, Hideyuki Komekado, Hiroshi Seno, Tsutomu Chiba

Backgrounds and aims Gastric cancer develops in the context of chronic inflammation ofgastric mucosa. Although our understanding of this disease has improved during the pastdecades, prognosis of patients with advanced gastric cancer remains poor. So, elucidatingunderlying molecular mechanisms is required to improve the prognosis of the patients.Nardilysin (N-arginine dibasic convertase; NRDc), a metalloendopeptidase of the M16 family,has been reported to promote the ectodomain shedding of precursor form of various growthfactors and cytokines via enhancing protease activities of a disintegrin and metalloproteinase(ADAM) proteins. Tumor necrosis factor (TNF)-α, one of the key inflammatory cytokines,is activated though the shedding of membrane-bound pro-TNF-α in an ADAM17/NRDc-dependent fashion. Here, we aimed to examine the growth-promoting role of NRDc ingastric cancer focusing on the activation of TNF-α and its downstream signaling cascades.Methods Serum NRDc concentration in gastric cancer patients was determined by ELISA.NRDc immunoreactivity was examined in surgically resected gastric cancer specimens. Afterknockdown (KD) of NRDc by RNAi, growth of human gastric cancer cell lines, cytokineexpression, and activation of various signaling cascades were analyzed both In Vitro and inxenograft experiments. Results In gastric cancer patients, serum NRDc concentration wassignificantly elevated. In surgically resected gastric cancer specimens, NRDc immunoreactivitywas detected in the cytoplasm of gastric cancer epithelium, primarily at the plasmamembrane.In gastric cancer cells, NRDc-knockdown (KD) suppressed cellular proliferation. Simultan-eously, NRDc-KD reduced ADAM17 protease activity and the ability to shed pro-TNF-α,and decreased the secretions of TNF-α and its downstream cytokines, such as IL-6. Nuclearextracts of NRDc-KD gastric cancer cells had attenuated DNA-binding activities for bothNF-κB and STAT3 consensus sequences. , and the expression of STAT3-regulated growth-promoting or anti-apoptotic genes, such as Cyclin D1, c-Myc, and Bcl-2, was also reduced.Treatment with either recombinant TNF-α or IL-6 protein restored the growth of NRDc-KDgastric cancer cells. In xenograft experiments, growth of gastric cancer cells was significantlysuppressed after KD of NRDc by RNAi. Conclusions Our results suggest that growth ofgastric cancer cells is maintained by autonomous TNF-α-NF-κB and IL-6-STAT3 signaling,and that NRDc activates these signaling cascades by stimulating ectodomain shedding of TNF-α and enhancing NF-κB-regulated cytokine expression. Suppression of NRDc expression byRNAi breaks these autonomous cytokine signaling cascades which are essential for growthof gastric cancer cells. Thus, NRDc may be a novel therapeutic target against gastric cancer.

953

Epigenetic Silencing of NR4A3 and LOXL4 by Aberrant JAK/STAT SignalingPredicts Prognosis in Gastric CancerLi-Han Zeng, Liang-Yu Chang, Jian-Liang Chou, Alfred S. Cheng, Kun-Tu Yeh, ClaudiaDittner, Jiayuh Lin, Michael W. Chan

Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlyingmechanism in the gastric carcinogenesis.Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathwayin gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothes-ized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 targetgenes in gastric cancer. To test this hypothesis, a constitutively activated mouse STAT3mutant (STAT3c) was transfected into MKN28 gastric cancer cells in which the JAK/STATsignaling pathway is inactive. STAT3c stable transfectant (S16) showing hyperphosphoryl-ation of STAT3 demonstrated increased cell proliferation as compared to vector control(C9). Integrative expression microarray coupled with bioinformatic analysis identified putat-ive STAT3 targets, NR4A3 and LOXL4 that are down-regulated in S16 cells. In associationwith up-regulation of DNMT1, NR4A3 and LOXL4 exhibited increased promoter methylationin S16 but not C9 cells as demonstrated by bisulphite sequencing and demethylationtreatment. Interestingly, NR4A3 was also found to be epigenetically silenced in AGS cellswhere JAK/STAT signaling is constitutively activated. ChIP-PCR experiment revealed thatSTAT3 bound to the putative STAT3 binding site in NR4A3 promoter of AGS cells. Depletionof STAT3 by lenti-virus infection restored NR4A3 expression in this cell. Interestingly,luciferase reporter assay using the NR4A3 promoter containing putative STAT3 binding siteexhibited a further 1.6 fold increment after deleting the STAT3 binding region (P<0.005).Ectopic expression of NR4A3 in AGS cells reduced cancer cell growth in colony formationassay (P<0.001). In clinical specimens, quantitative MSP demonstrated a significant correla-tion between the degree of NR4A3 methylation and STAT3 nuclear translocation in 72 gastrictumor samples (P<0.05). Importantly, methylation of NR4A3 was significantly associatedwith patients with shorter survival (P<0.05). In contrast, IM patient (n=66) demonstrateda significantly higher methylation of LOXL4 than in tumor (n=66) and gastritis (n=9) samples(P<0.0005). IM patients infected with H. pylori have higher LOXL4 methylation than thatwithout H. pylori. Surprisingly, multivariate COX-regression analysis showed that LOXL4methylation is an independent prognostic marker and is associated with better prognosisin gastric cancer patients (HR=0.467; 95% CI: 0.248-0.877; P=0.018). In conclusion, ourresult demonstrated that aberrant JAK/STAT3 signaling induces epigenetic silencing ofNR4A3and LOXL4 in gastric cancer. Methylation of NR4A3 and LOXL4 predicts survival in gastriccancer patients.

S-164AGA Abstracts

954

Rational Effectiveness of Anti-IL-6 Receptor Neutralizing Antibody andNOTCH3 Inhibition in Combination Chemotherapy Targeting Cancer StemCellYing Jin, Masahiko Tsujii, Jumpei Kondo, Takuya Inoue, Motohiko Kato, ShunsukeYamamoto, Yoshito Hayashi, Tomofumi Akasaka, Takuya Yamada, Eri Shiraishi, TakahiroInoue, Akira Mukai, Satoshi Hiyama, Sachiko Nakajima, Shinichiro Shinzaki, TsutomuNishida, Kenji Watabe, Hideki Iijima, Tetsuo Takehara

Background and aims: Recent studies have shown that cancer stem cell (CSC) can initiateand sustain tumor growth, and exhibit resistance to clinical cytotoxic therapies. Therefore,CSCs are thought to be the main potential target for anticancer therapy. Interleukin-6 (IL-6) promotes proliferation and drug resistance in colorectal carcinoma levels and its serumlevels are correlated with survival. Therefore, IL-6 and its downstream molecule, SignalTransducers and Activators of Transcription 3 (Stat3), are expected as molecular targets.Other roles are, however, reported: IL-6 induces differentiation and reduces malignantfeatures. Aims: In the present study, we investigated the effect of IL-6 on stem cell biologyand chemo-resistance of colon cancer cells and explored potential molecular targets. Methods:WiDr, a colon cancer cell line, was cultured in serum free non-adherent, three dimensionedspheroid-forming condition to enrich in stem cells. The expressions of stem cell-related genes(oct-4, klf-4, Bmi1, and Lgr5) and Notch3, and STAT3 activation (STAT3 phosphorylation(STAT3p)) were investigated. The effects of STAT3 inhibitors and anti-IL-6 receptor neutraliz-ing antibody (IL-6 Ab) on expression of stem-related genes, cell proliferation, and chemores-istance to 5-fluorouracil (5FU) were determined. Results: Spheroid forming WiDr cells wereexpressing higher levels of oct-4, klf4, Bmi1, Lgr5, Notch3 and IL-6 and STAT3p. IL-6treatment enhanced STAT3p, expression of oct-4, Notch3, and Lgr5, and chemoresistanceto 5FU in spheroid forming WiDr cells. IL-6 Ab significantly reduced STAT3p and oct-4,Notch3, and Lgr5 expression, proliferation, and 5FU chemoresistance, whereas STAT3inhibitors reduced proliferation but enhanced Notch3 and oct-4 expression and 5FU chemor-esistance. siRNA for Notch3 suppressed expression of oct-4 and Lgr5, spheroid formationand 5FU chemoresistance. Conclusion: These results indicated that IL-6 functioned indichotomous pathways: activation of STAT3 and Notch3 induction, the former was leadingto accelerated proliferation but reduced chemoresistance, and the latter was involved incancer stem cell biology and enhanced chemoresistance. It was suggested that anti-IL-6receptor neutralizing antibody or Notch3 inhibition was better than STAT3 inhibitors forCSC-targeting concomitant therapy with anticancer drugs.

956

The Wnt Signaling Pathway is Deregulated in Human Neuroendocrine TumorCells by Epigenetic Silencing of Sfrp-1 and WIF-1Ji Tae Kim, Jing Li, Eun Ryoung Jang, Piotr Rychahou, Heidi L. Weiss, Courtney M.Townsend, Chunming Liu, B. Mark Evers

Accumulation of β-catenin in the cytoplasm and/or nucleus has been observed in gastrointesti-nal (GI) carcinoid tumors. However, mutation and altered expression of Wnt pathwaycomponents have not been extensively investigated in neuroendocrine tumors (NETs). Previ-ously, we identified a missense (E1317Q) and a silent (T1493T) mutation of the APC inhuman NET cells. In addition, we showed that transcriptional expression of Wnt inhibitors,Axin-2, DKK-1, DKK-3, and APC was silenced by methylation of the promoter or histonein NETs. Here, we examined: i) the transcriptional status of other negative regulators of theWnt pathway, SFRP-1 and WIF-1, ii) the mechanism of aberrant repression of these genesin NET cells, and iii) the functional validation of these genes in NET cells by overexpression.METHODS. i) Four human NET cell lines including BON (pancreatic carcinoid), QGP-1(pancreatic somatostatinoma), NCI-H727 (bronchial carcinoid) and UMC-11 (bronchialcarcinoid) were examined. Gel based RT-PCR and qRT-PCR were performed to assessrestoration of SFRP-1 and WIF-1 in BON cells treated with a demethylating agent, 5-aza-2'-deoxycytidine (5-aza-CdR). ii) Methylation-specific PCR (MSP) and bisulfite sequencingwere used to determine CpG island promoter methylation of the above genes. Chromatinimmunoprecipitation (ChIP) assays were performed to determine whether levels of histoneH3 Lys9 di-methylation (H3K9me2), which is implicated in DNA-methylation associatedgene silencing, were regulated by 5-aza-CdR in the unmethylated gene promoter. iii) Toconfirm tumor suppressor functions of SFRP-1 and WIF-1 In Vitro and In Vivo, we analyzedthe transcriptional activity of the Wnt pathway, colony formation, anchorage-independentgrowth and tumor growth in nude mice using BON cells overexpressing these genes.RESULTS. Treatment of 5-aza-CdR induced the restoration of SFRP-1 and WIF-1 mRNAexpression in BON cells. MSP and bisulfite sequencing of these genes demonstrated thatthe promoter of SFRP-1 was methylated in the four cells and NET clinical samples, includingfour GI carcinoids, whereas the promoter of WIF-1 was unmethylated. Levels of H3K9me2in theWIF-1 promoter were attenuated by 5-aza-CdR in a dose-dependent manner. Transientoverexpression of these Wnt inhibitory genes in BON cells decreased transcriptional activityof the Wnt pathway, and colony formation was attenuated compared with control cells. Inaddition, stable restoration of SFRP-1 or WIF-1 in BON cells resulted in suppression ofanchorage-independent growth and inhibition of tumor growth in nude mice. CONCLU-SIONS. Our findings suggest that activation of theWnt signaling pathway, through epigeneticsilencing of SFRP-1 and WIF-1, may contribute to the pathogenesis of GI NETs and thatWnt inhibitors, as tumor suppressor proteins, may be utilized for potential therapeutictargets against NETs.