Mo1659 The PI3K/AKT/mTOR Pathway in Gastric Cancer and Its Potential Relationship With Mir-125b,...
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Mo1658 Acid-Induced Decrease in Cell Apoptosis Is Mediated by Activation of NADPH Oxidase NOX5-S, NF-κB and Sodd in Barrett's Esophageal Adenocarcinoma Cells Jie Hong, Dan Li, Weibiao Cao Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood. Acid reflux may play an important role in this progression. We have previously shown that acid exposure increases H 2 O 2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-depen- dent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers. In this study we examined the role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD. SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T. Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells. Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immuno- precipitation (ChIP) assay showed that SODD DNA was detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT. Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleo- tide significantly reduced binding. The addition of the mutant SODD oligonucleotide had less effect on binding. We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD. Supported by NIH NIDDK R01 DK080703. Mo1659 The PI3K/AKT/mTOR Pathway in Gastric Cancer and Its Potential Relationship With Mir-125b, Mir-451 and Mir-101 Ismael Riquelme, Oscar Tapia, Alejandra M. Sandoval, Pamela Leal, Pablo Letelier, Juan C. Roa Introduction: Gastric cancer (GC) is associated with aberrant expression of miRNAs and deregulation in signaling pathways. We performed in silico predictive analyses to know which miRNAs could be regulating the PI3K/AKT/mTOR pathway and could play an important role in the development of GC. We found that miR-125b, miR-451 and miR-101 could target the mRNA of PI3K and AKT (miR-125b), TSC1 (miR-451), and mTOR and PI3K (miR- 101), all members of this pathway. The aim of this study was to evaluate the transcriptional and protein expression of the most important components of PI3K/AKT/mTOR pathway in tissues and GC cell lines and associate their expression with the expression of miR-125b, miR-451 and miR-101. Methods: The expressions of PI3K, AKT, PTEN, TSC1, mTOR, P70S6K1, 4E-BP1 and eIF4E genes and expressions of miR-125b, miR-451 and miR-101 were analyzed in 25 advanced GC tissues and their 25 paired non-tumor tissues (NT), and in AGS, MKN28 and MKN45 GC cell lines by qRT-PCR. The protein expression of PI3K/ AKT/mTOR was performed by IHC in TMAs of 142 gastric tissue samples (71 advanced GC and 71 paired non-tumor tissues). The proteins studied were PI3K, PTEN, AKT, phospho- AKT, mTOR, phospho-mTOR, p70S6K1, phospho-p70S6K1, 4E-BP1, phospho-4E-BP1, eIF4E and phospho-eIF4E. Clinical and pathological data of patients was used. Results: Increased protein expression was found by IHC for PI3K, AKT, phospho-AKT, phospho- mTOR, p70S6K, phospho-p70S6K, phospho-4E-BP1, eIF4E and phospho-eIF4E in tumor tissues compared with non-tumor tissues (p<0,05). Conversely, levels of PTEN were higher in non-tumor tissues (p <0.001). Moreover, Kaplan-Meier analysis showed a poor survival in those patients with low expression of 4E-BP1 (p=0,03). The gene expression of PI3K, AKT, mTOR, P70S6K1 y eIF4E in AGS, MKN28 and MKN45 cell lines was significantly higher compared with non-tumor cells. PTEN was downregulated in these 3 cell lines. Decreased expressions of miR-125b (p=0,039), miR-451 (p=0,0013) and miR-101 (p= 0,0005) were found in GC tissues compared with NT. Also, lower expressions of miR-451 and miR-101 were found in the three GC cell lines (p<0,0001 for all). Meanwhile, miR- 125b was found decreased only in AGS and MKN45 cell lines (p<0,0001 both). Conclusion: This study has shown that PI3K/AKT/mTOR pathway is activated in tumor tissues of GC and these data could help us to validate this pathway as a potential therapeutic target for this malignancy. Moreover, miR-125b, miR-451 and miR-101 have association with the expression of some members of the PI3K/AKT/mTOR pathway. This relationship could be important in order to modulate the behavior of this pathway during carcinogenesis. However, further studies are necessary to confirm the direct interplay between these miRNAs and the PI3K/AKT/mTOR. Grant support: FONDECYT #11110239, CEGIN #09CN14-5960, CONICYT #24121456 Mo1660 FOXP3 Inhibits Cell Proliferation of Gastric Cancer Cells Through Modulation of the TGF-β/SMAD Signaling Pathway Liu Yimei, Ma Guifen, Shiyao Chen Our previews studies implied Forkhead Box Protein (FoxP3) induce cell apoptosis by apoptosis signaling pathway and inhibit proliferation of gastric cancer (GC) cells. However, the mechanism of decreased cell proliferation is still unclear. We examined whether FoxP3 affects human GC cell (AGS) proliferation by activation of TGF-β/Smad signaling pathway. S-629 AGA Abstracts AGS were treated with either up-regulating or down-regulating FoxP3 gene. We determined cell proliferation, nuclear translocation of Smad2/3, expression of Smad2/3, p21 and forma- tion of Smad2/3-Smad4 complex by multiple cellular and molecular approaches, such as CCK-8 assay, EdU assay, immunofluorescence, Immunoprecipitation, Western Blot and quantities polymerase chain reaction (PCR). We demonstrated that FoxP3 over-expression inhibited cell proliferation and silencing of FoxP3 by siRNA promote AGS cells growth. FoxP3 over-expression activated Smad2/3 phosphorylation, increased nuclear translocation of Smad protein, expression of p21 and Smad2/3-Smad4 complex formation in AGS cells. Furthermore, we found that down-regulating of FoxP3 surpressed Smad2/3 phosphorylation, expression of p21 and Smad2/3-Smad4 complex formation in AGS cells. These data demon- strated thatFoxP3 inhibit the growth of gastric cancer cells, which may be related to the TGF/Smad signal. Overexpression of FoxP3 inhibits the cell growth FoxP3 induces the expression of phosphorylation-Smad2/3 and Smad2/3-Smad4 complex proteins Mo1661 Comprehensive DNA Methylation and Extensive Mutation Analysis of Gastric Neuroendocrine Carcinoma Takayuki Ando, Ayumu Hosokawa, Sohachi Nanjo, Yuko Ueda, Momoko Akashi, Akira Ueda, Hiroshi Mihara, Shinya Kajiura, Haruka Fujinami, Jun Nishikawa, Toshiro Sugiyama OBJECTIVE: Neuroendocrine cancer (NEC) of the stomach is a rare type of cancer that is often diagnosed at an advanced stage. The molecular characterization of NEC cells is poorly understood, although the gastrointestinal NEC shares genetic alterations with adenocarci- noma and shows non-neuroendocrine cell lineage in limited small studies. In this study, we aimed to clarify the molecular characterization of gastric NEC and to find NEC-specific alterations in comparison with adenocarcinoma. METHODS: Twenty samples from gastric cancers with advanced stage (11 adenocarcinomas and 9 NECs) and paired non-cancerous mucosa were obtained by endoscopic biopsy. Analysis of DNA methylation was performed using the Infinium HumanMethylation 450 BeadChip array, covering 482,421 CpG sites (Illumina), and analysis of sequence variations of 55 cancer-related genes was performed using the Ion AmpliSeq Panel Kit (Life Technologies). DNA methylation and sequencing variation was confirmed using methylation-specific PCR and dideoxy sequencing. RESULTS: Comprehensive methylation analysis of 20 gastric cancers revealed that the number of aberrantly methylated genes was highly variable between individual gastric NEC and adeno- carcinoma. Among the hundreds of tumor-specific methylations discovered, 15 genes with promoter methylation in >30% of NECs and 0% of adenocarcinomas were identified. Among the genes with NEC-specific methylation, DLL1 is well-known gene to be involved in AGA Abstracts
Transcript of Mo1659 The PI3K/AKT/mTOR Pathway in Gastric Cancer and Its Potential Relationship With Mir-125b,...
Mo1658
Acid-Induced Decrease in Cell Apoptosis Is Mediated by Activation of NADPHOxidase NOX5-S, NF-κB and Sodd in Barrett's Esophageal AdenocarcinomaCellsJie Hong, Dan Li, Weibiao Cao
Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA)are not fully understood. Acid reflux may play an important role in this progression. Wehave previously shown that acid exposure increases H2O2 production and cell proliferation,and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidaseNOX5-S. However, which downstream proteins mediate acid-induced and NOX5-S-depen-dent decrease in cell apoptosis are not fully understood. Silencer of death domain (SODD),also known as BCL2-associated athanogene 4, is known to be involved in the survival ofcancer cells and related to aggressiveness of different cancers. In this study we examinedthe role of SODD in acid-induced decrease in cell apoptosis. Human genomic microarrayanalysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decreasein the expression of SODD. SODD mRNA levels were significantly higher in EA cell lineFLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett'scell line BAR-T. Acid treatment significantly upregulated SODD expression and increasedSODD promoter activity in FLO cells. Acid-induced increase in SODD expression andpromoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1p50 in FLO cells. One potential NF-κB binding element was identified in the DNMT1promoter region: GGGACACCCT (positions -310 to -300 from ATG). Chromatin immuno-precipitation (ChIP) assay showed that SODD DNA was detectable in the immunoprecipitatedchromatin samples of FLO cell lysate by PCR using a pair of primers covering the abovepotential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter. Thegel shift mobility assays showed that one prominent complex was detectable with IRdye700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT.Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleo-tide significantly reduced binding. The addition of the mutant SODD oligonucleotide hadless effect on binding. We conclude that acid-induced decrease in cell apoptosis may dependon sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells. NF-κB1 p50may directly bind to the SODD promoter and promote the expression of SODD. Supportedby NIH NIDDK R01 DK080703.
Mo1659
The PI3K/AKT/mTOR Pathway in Gastric Cancer and Its PotentialRelationship With Mir-125b, Mir-451 and Mir-101Ismael Riquelme, Oscar Tapia, Alejandra M. Sandoval, Pamela Leal, Pablo Letelier, JuanC. Roa
Introduction: Gastric cancer (GC) is associated with aberrant expression of miRNAs andderegulation in signaling pathways. We performed in silico predictive analyses to know whichmiRNAs could be regulating the PI3K/AKT/mTOR pathway and could play an important rolein the development of GC. We found that miR-125b, miR-451 and miR-101 could targetthe mRNA of PI3K and AKT (miR-125b), TSC1 (miR-451), and mTOR and PI3K (miR-101), all members of this pathway. The aim of this study was to evaluate the transcriptionaland protein expression of the most important components of PI3K/AKT/mTOR pathway intissues and GC cell lines and associate their expression with the expression of miR-125b,miR-451 and miR-101. Methods: The expressions of PI3K, AKT, PTEN, TSC1, mTOR,P70S6K1, 4E-BP1 and eIF4E genes and expressions of miR-125b, miR-451 and miR-101were analyzed in 25 advanced GC tissues and their 25 paired non-tumor tissues (NT), andin AGS, MKN28 and MKN45 GC cell lines by qRT-PCR. The protein expression of PI3K/AKT/mTOR was performed by IHC in TMAs of 142 gastric tissue samples (71 advancedGC and 71 paired non-tumor tissues). The proteins studied were PI3K, PTEN, AKT, phospho-AKT, mTOR, phospho-mTOR, p70S6K1, phospho-p70S6K1, 4E-BP1, phospho-4E-BP1,eIF4E and phospho-eIF4E. Clinical and pathological data of patients was used. Results:Increased protein expression was found by IHC for PI3K, AKT, phospho-AKT, phospho-mTOR, p70S6K, phospho-p70S6K, phospho-4E-BP1, eIF4E and phospho-eIF4E in tumortissues compared with non-tumor tissues (p<0,05). Conversely, levels of PTEN were higherin non-tumor tissues (p <0.001). Moreover, Kaplan-Meier analysis showed a poor survivalin those patients with low expression of 4E-BP1 (p=0,03). The gene expression of PI3K,AKT, mTOR, P70S6K1 y eIF4E in AGS, MKN28 and MKN45 cell lines was significantlyhigher compared with non-tumor cells. PTEN was downregulated in these 3 cell lines.Decreased expressions of miR-125b (p=0,039), miR-451 (p=0,0013) and miR-101 (p=0,0005) were found in GC tissues compared with NT. Also, lower expressions of miR-451and miR-101 were found in the three GC cell lines (p<0,0001 for all). Meanwhile, miR-125b was found decreased only in AGS and MKN45 cell lines (p<0,0001 both). Conclusion:This study has shown that PI3K/AKT/mTOR pathway is activated in tumor tissues of GCand these data could help us to validate this pathway as a potential therapeutic target forthis malignancy. Moreover, miR-125b, miR-451 and miR-101 have association with theexpression of some members of the PI3K/AKT/mTOR pathway. This relationship could beimportant in order to modulate the behavior of this pathway during carcinogenesis. However,further studies are necessary to confirm the direct interplay between these miRNAs andthe PI3K/AKT/mTOR. Grant support: FONDECYT #11110239, CEGIN #09CN14-5960,CONICYT #24121456
Mo1660
FOXP3 Inhibits Cell Proliferation of Gastric Cancer Cells Through Modulationof the TGF-β/SMAD Signaling PathwayLiu Yimei, Ma Guifen, Shiyao Chen
Our previews studies implied Forkhead Box Protein (FoxP3) induce cell apoptosis byapoptosis signaling pathway and inhibit proliferation of gastric cancer (GC) cells. However,the mechanism of decreased cell proliferation is still unclear. We examined whether FoxP3affects human GC cell (AGS) proliferation by activation of TGF-β/Smad signaling pathway.
S-629 AGA Abstracts
AGS were treated with either up-regulating or down-regulating FoxP3 gene. We determinedcell proliferation, nuclear translocation of Smad2/3, expression of Smad2/3, p21 and forma-tion of Smad2/3-Smad4 complex by multiple cellular and molecular approaches, such asCCK-8 assay, EdU assay, immunofluorescence, Immunoprecipitation, Western Blot andquantities polymerase chain reaction (PCR). We demonstrated that FoxP3 over-expressioninhibited cell proliferation and silencing of FoxP3 by siRNA promote AGS cells growth.FoxP3 over-expression activated Smad2/3 phosphorylation, increased nuclear translocationof Smad protein, expression of p21 and Smad2/3-Smad4 complex formation in AGS cells.Furthermore, we found that down-regulating of FoxP3 surpressed Smad2/3 phosphorylation,expression of p21 and Smad2/3-Smad4 complex formation in AGS cells. These data demon-strated thatFoxP3 inhibit the growth of gastric cancer cells, which may be related to theTGF/Smad signal.
Overexpression of FoxP3 inhibits the cell growth
FoxP3 induces the expression of phosphorylation-Smad2/3 and Smad2/3-Smad4 complexproteins
Mo1661
Comprehensive DNA Methylation and Extensive Mutation Analysis of GastricNeuroendocrine CarcinomaTakayuki Ando, Ayumu Hosokawa, Sohachi Nanjo, Yuko Ueda, Momoko Akashi, AkiraUeda, Hiroshi Mihara, Shinya Kajiura, Haruka Fujinami, Jun Nishikawa, ToshiroSugiyama
OBJECTIVE: Neuroendocrine cancer (NEC) of the stomach is a rare type of cancer that isoften diagnosed at an advanced stage. The molecular characterization of NEC cells is poorlyunderstood, although the gastrointestinal NEC shares genetic alterations with adenocarci-noma and shows non-neuroendocrine cell lineage in limited small studies. In this study,we aimed to clarify the molecular characterization of gastric NEC and to find NEC-specificalterations in comparison with adenocarcinoma. METHODS: Twenty samples from gastriccancers with advanced stage (11 adenocarcinomas and 9 NECs) and paired non-cancerousmucosa were obtained by endoscopic biopsy. Analysis of DNA methylation was performedusing the Infinium HumanMethylation 450 BeadChip array, covering 482,421 CpG sites(Illumina), and analysis of sequence variations of 55 cancer-related genes was performedusing the Ion AmpliSeq Panel Kit (Life Technologies). DNA methylation and sequencingvariation was confirmed using methylation-specific PCR and dideoxy sequencing. RESULTS:Comprehensive methylation analysis of 20 gastric cancers revealed that the number ofaberrantly methylated genes was highly variable between individual gastric NEC and adeno-carcinoma. Among the hundreds of tumor-specific methylations discovered, 15 genes withpromoter methylation in >30% of NECs and 0% of adenocarcinomas were identified. Amongthe genes with NEC-specific methylation, DLL1 is well-known gene to be involved in
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