Tu1608

Chemical Inhibitors of the Wnt Pathway or G9a Showed Anti-Tumor Effectsin Human Neuroendocrine Tumor Cells With Deregulated Wnt/β-CateninSignalingJi Tae Kim, Jing Li, Eun Ryoung Jang, Dana L. Napier, Heidi L. Weiss, Eun Lee, CourtneyM. Townsend, Chunming Liu, B. Mark Evers

Carcinoid tumors are relatively uncommon neuroendocrine tumors (NETs) that are increasingin incidence. Mutation and altered expression of Wnt/β-catenin signaling components havebeen described in many tumors but have not been well-studied in NETs. Previously weidentified a missense (E1317Q) and a silent (T1493T) mutation of the APC gene in humanNET cell lines. In addition, we demonstrated that transcriptional expression of Wnt inhibitorswas silenced by promoter methylation (SFRP-1 and Axin-2) or histone modification,H3K9me2 (DKK-1, DKK-3 andWIF-1) in the cells. Furthermore, we showed that restorationof the Wnt inhibitory genes resulted in tumor suppressor functions. Here, we examined: i)accumulation of β-catenin in the cytoplasm and/or nucleus in clinical NET tissues, ii)mutations of β-catenin and APC genes and promoter methylation status of SFRP-1 andAxin-2 in the NET tissues, and iii) anti-tumor effects of the chemical inhibitors for Wnt/ β-catenin pathway or G9a in NET cell lines. METHODS. i) Immunohistochemical localizationof β-catenin was examined in clinical carcinoid tissues from colon (n=1), small bowel (n=11), lung (n=6) and thymus (n=2). ii) Mutations of β-catenin (exon 3) and APC (exon15) in the tested carcinoid tissues were analyzed by PCR and direct sequencing analysis.Methylation-specific PCR was used to determine CpG island methylation of SFRP-1 andAxin-2 in the clinical tissues. iii) To determine anti-tumor effects of Wnt inhibitors (iCRT3and novel FIDAS compounds) or a G9a inhibitor (UNC0638), we analyzed various cellulareffects such as cell proliferation and expression level of the Wnt pathway related proteinsin human NET cell lines BON and QGP-1. RESULTS. We observed accumulation of β-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutationalanalysis, the mutations of β-catenin (I35S) and APC (T1493T) were identified in the NETtissues. Specifically, aberrant methylation of SFRP-1 was observed in the majority of theclinical NET tissues. Additionally, the chemical inhibitors for Wnt/ β-catenin signaling orG9a suppressed cell proliferation, TCF/LEF-responsive reporter activity and levels of c-Myc(a target of Wnt/β-catenin pathway) and increased sensitivity to serum-starvation inducedapoptosis. CONCLUSIONS. Our findings suggest that aberrant Wnt/ β-catenin signaling,through mutations of β-catenin or APC or CpG island methylation of SFRP-1, contributesto the pathogenesis and growth of clinical NETs and that the chemical inhibitors for Wnt/β-catenin signaling or G9a may be utilized as potential therapeutic agents against NETs.

Tu1609

The Methylation Status of Human DNA Determines the Autolog Activation ofTLR9 Pathway on HT29 Colorectal Cancer CellsIstván Furi, Sandor Spisak, Árpád V. Patai, Ferenc Sipos, Györgyi Muzes, AlexandraKalmár, Gergo Kiszner, Gabor Valcz, Barnabás Wichmann, Béla Molnár, Zsolt Tulassay

Background: Toll-like receptor 9 (TLR9) recognises cytosin-guanine dinucleotides frombacteria, fungi, prokaryotes and viruses and synthetic oligodeoxynucleotide (ODN)sequences. Till today no evidence exists, that the human DNA released thorough apoptosis,necrosis can act through TLR9 signaling in a paracrine way. Aims: Examine the geneexpression level changes of TLR9 and genes on TLR9 signal transduction pathway on themRNA level after the treatment by unmethylated DNA, and methylated DNA isolated fromHT29 cells on HT29cells. To analyse the expression of DNA methyltransferase expressionchanges before and after DNA treatment. Materials and methods: Genomic DNA for thetreatment was isolated from HT29 cells and partially consequtively methylated by NewEngland Biolabs CpG Methyltransferase (M.SssI). Subsequently, 0.5x106 HT29 cells weretreated with 15μg of DNA in 2 ml RPMI 1640 without BSA for 6 hours. The cells wereharvested and total RNA was isolated both before and after DNA treatment using QiagenRNeasy Mini Kit. Expression levels of genes were examined by RT-PCR for IL-8, MYD88A,NFκB, TLR9 RP 11, TRAF6, Immunocytochemistry was performed for TLR9, DNA methyl-transferases (DNMT1, DNMT3a, DNMT3b), proliferation and differentiation factors (CDX2,CK ). At immunocytochemistry the cells from DNA treated and untreated samples weredivided into 4 groups (strongly positive, meanly positive, weakly positive and negative).Results: TLR RP 11 level was significantly increased after the treatment by non-methylatedDNA, (p,0.05). MYD88A, TRAF6, IRAK2 genes showed significant overexpression at thetreatment by the non-methylated DNA (p,0.05). Treatment by methylated DNA showedsignificant proinflammatory response mediated by high level of NFκB, IRAK2, IL-8 (p,0.05).CK expression significantly increased for DNA treatment (32,2%, 17,4%, 46,1%, 4,3%) ascompared to the control(0,0%, 3,4%, 36,8%, 59,8%) (p,=0,0001). DNAmethylation activitycould be induced by DNA treatment as it was shown by DNMT3a IHC (0,0%, 0,0%,17,5%, 82,5%) as compared to untreated control(0,0%, 0,0%, 1,8%, 98,2%) (p ,=0,0001).Conclusions: Human DNA can react on paracrine way on cancer cells through TLR9 activa-tion. DNA treatment resulted significant overexpression of CK and DNMT3a. The DNAtreatment increased the differentation and the methyltransferase activity. The methylationrate of the released DNA is reflected in the cell's response. Released human DNA is notonly a biomarker for cancer detection, but also biologically active molecule in a local context.

Tu1610

Characterization of Small Bowel Adenocarcinoma Cell Line and Evaluation ofAnti-Cancer Drug Efficacy Against Small Bowel AdenocarcinomaHirobumi Suzuki, Yoshihiro Hirata, Souzaburo Ihara, Kosuke Sakitani, Yuka Kobayashi,Atsuo Yamada, Yutaka Yamaji, Kazuhiko Koike

Introduction; Small bowel adenocarcinoma (SBA) is rare malignancy. The rarity has madeit difficult to unravel the pathogenesis and develop the treatment. To understand molecularcharacteristics and develop new treatment of SBA, we established an SBA cell line. Materialsand Methods; An SBA cell line (SIAC cell) was established from a biopsy specimen ofjejunum SBA. Features of SIAC cell were investigated by western blot, DNA sequence

S-805 AGA Abstracts

for replication errors (RERs), immunohistochemistry, immunocytochemistry, and luciferaseassay. 9 clinical SBA samples were also analyzed by DNA sequence for RERs and immunohis-tochemistry of mismatch repair (MMR) protein. Efficacy of drug candidates against SBA wasevaluated with cell proliferative assay of SIAC cells. Results; SIAC cell had no mutations inKRAS, BRAF, and APC, but had heterozygous β-catenin deletion mutation which lacks 11-137 amino acids. Luciferase assay showed the enhanced Wnt/ β-catenin pathway. Treatmentwith protein synthesis inhibitor, cycloheximide, indicated that β-catenin deletion mutantwas more stable than wild type. SIAC cell lacked MLH1 and MSH6 expression, and showeddecreased MSH2 expression. In addition, MMR target genes such as TGF βRII, ACVRII, BAX,and MSH6 showed flameshift mutations. Of 9 SBA clinical samples, 2(22%) had large-scaleN-terminal deletions in β-catenin. The loss or attenuation of expression of MLH1, MSH2and MSH6 in immunohistochemistry was identified in 4/9(44%), 2/9(22%), 3/9(33%),respectively. Frequency of MMR target gene mutations were as follow; TGF βRII(9/9),IGFIIR(0/9), BAX(2/9),MSH6(4/9),ACVRII(1/9). Cell proliferative assay using 140 com-pounds including molecular targeting drugs showed that Bortezomib and Eribulin inhibitSIAC cell growth at the optimum concentration. Conclusion; An established SBA cell lineshows similar molecular disorders observed in clinical SBA samples, i.e. β-catenin deletionand MMR protein deficiency, suggesting that this cell line is a useful tool for SBA research.Bortezomib and Eribulin may be good candidates for SBA treatment.

Tu1611

The Expression and Function of Toll-Like Receptors 3 and 9 in Human ColonCarcinomasMasahiko Tameda, Katsuya Shiraki, Kazushi Sugimoto, Keiichiro Nojiri, SatokoKusagawa, Yuji Inagaki, Suguru Ogura, Norihiko Yamamoto, Chika Kasai, HidekazuInoue, Yoshiyuki Takei, Masaaki Ito, Koujirou Takase

Background & Aims: Toll-like receptors (TLRs) are pattern-recognition receptors that areimportant in immune signaling. TLR recognition of various viral components includingdouble-stranded RNA (TLR3) and unmethylated CpG-DNA (TLR9) plays a crucial role incell survival. However, TLR expression and function in colon carcinoma cells are not wellclarified Methods: We investigated the expression of TLR3 and TLR9 in colon carcinomacells using immunohistochemical methods. The function of TLR3 and TLR9 signaling incarcinoma cell lines was studied by direct cell stimulation with, or by cell transfection of,polyinosinic-polycytidylic acid (Poly I:C), a synthetic form of dsRNA, and by cell stimulationwith CpG-oligodeoxynucleotides (ODNs), respectively. Results: Positive TLR3 and TLR9immunohistochemical staining was observed in 91% and 86% of human hepatocellularcarcinoma (HCC) tissues, respectively. Cell surface stimulation of TLR3 with Poly I:C didnot affect cell viability but did activate NF-κB activity. In contrast, stimulation of intracellularTLRs with transfected Poly I:C significantly induced apoptosis. Cell surface stimulation ofTLR9 with CpG-ODNs promoted cell proliferation, and, furthermore, these CpG-ODN TLR9agonists reduced the cytotoxicity of the anti-cancer drug Adriamycin. Conclusions: Cellsurface expression of TLR3 and TLR9 in colon carcinoma cells plays an important role incell survival. In addition, the proapoptotic activity of intracellularly expressed TLR3 mayprovide the possibility of using TLR3 agonists as novel clinical cytotoxic agents against coloncarcinoma cells.

Tu1612

Reprimo, a p53 Dependent G2 Arrest Mediator, Is Downregulated in theSignet-Ring Cell Type, a Particularly Aggressive Subset of Gastric CancerKathleen Saavedra, Wilda Olivares, Alejandra Sandoval, Benjamin García, Javier Cerda-Infante, Juan C. Roa, Alejandro H. Corvalan

Introduction: Gastric cancer (GC) is the second leading cause of cancer-related deathsworldwide and the first cause of death among Latin American countries, including Chile.Reprimo (RPRM), a p53 dependent G2 arrest mediator, has been identified as a putativetumor suppressor gene in a variety of tumors, including GC. In this study, we examinedthe immunohistochemical expression of RPRM in a large cohort of GC and correlatedthese results with clinicopathological parameters and patient survival. Method: A previouslyreported TissueMicroarray (TMA) platform (Clin Cancer Res 2012;16:3253-3259) containingtriplicate cores from 151 consecutive patients who had undergone surgery in a singleinstitution for histologically proven GC were selected for this study. RPRM and p53 proteinswere detected by immunohistochemistry (IHC) and percentage and staining intensity ofpositive tumor cells were assessed. The percentage of positive cells between 0 and 10% wasconsidered negative and more than 10% positive cells was considered positive. Immunohisto-chemical results were correlated with clinicopathological parameters and p53 expression(available for 49 cases). Cumulative overall survival rates were estimated using the Kaplan-Meier method, and survival differences were analyzed by the log-rank test. Results: Loss ofRPRM protein expression was found in 47% of GC cases, significantly associated to signet-ring cell-type histology (P , 0,05). Patients with expression of RPRM tended to have lowersurvival rates. No statistical significance was found between p53 and RPRM expression.Dis-cussion: Our findings suggests that RPRM is downregulated in a particularly aggressivesubset of gastric adenocarcinomas, the signet-ring cell type. Interestingly, patients whosetumors express this putative tumor suppressor gene tend to have a worse prognosis. Nocorrelation was observed between the expression of p53 and RPRM. This finding suggeststhe existence of other regulatory mechanisms of RPRM. Further studies are needed todetermine these regulatory mechanisms. Grant Support: Fondecyt #1111014, Fonde-f#D09I1137 from de Government of Chile to AHC.

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