Target Genes In Gastric Cancer Research Articles

Long non-coding RNAs as potential biomarkers or therapeutic targets in gastric cancer.

This study aimed to find lncRNAs and mRNAs that were expressed differently by combining microarray datasets from different studies. This was done to find important target genes in gastric cancer for anti-cancer therapy. Gastric cancer (GC) is the fourth most frequent and second-most deadly malignancy worldwide. Thus, genetic diagnosis and treatment should focus on genetic and epigenetic variables. Based on several studies, disordered expression of non-coding RNAs (ncRNAs), such as lncRNAs, regulate gastric cancer invasion and metastasis. Besides, lncRNAs cooperatively regulate gene expression and GC progression. We obtained differentially expressed mRNAs (DEmRNAs) and lncRNAs (DElncRNAs) from three GC tissue microarray datasets by meta-analysis and screened genes using the "Limma" package. Then, using the RNAInter database, we allocated DEmRNAs to each DElncRNA. ClusterProfiler and GOplot programs were used to analyze function enrichment pathways and gene ontologies for final DEmRNAs. A total of 9 differentially expressed lncRNAs (DElncRNAs) (5 up-regulated and 4 down-regulated), and 856 DEmRNAs (451 up-regulated and 405 down-regulated) between tumor and adjacent normal samples were found. Finally, 117 differentially expressed mRNAs were predicted as interactors of six DElncRNAs (H19, WT1-AS, EMX2OS, HOTAIR, ZEB1-AS1, and LINC00261). In order to promote cancer therapeutics and give knowledge on the process of carcinogenesis, our study projected a network of drug-gene interactions for discovered genes and presented relevant prospective biomarkers for the prognosis of patients with stomach cancer.

MicroRNA-30a arbitrates intestinal-type early gastric carcinogenesis by directly targeting ITGA2.

Spasmolytic polypeptide-expressing metaplasia (SPEM) is considered a precursor lesion of intestinal metaplasia and intestinal-type gastric cancer (GC), but little is known about microRNA alterations during metaplasia and GC developments. Here, we investigate miR-30a expression in gastric lesions and identify its novel target gene which is associated with the intestinal-type GC. We conducted in situ hybridization and qRT-PCR to determine miR-30a expression in gastric tissues. miR-30a functions were determined through induction or inhibition of miR-30a in GC cell lines. A gene microarray was utilized to confirm miR-30a target genes in GC, and siRNA-mediated target gene suppression and immunostaining were performed. The Cancer Genome Atlas data were utilized to validate gene expressions. We found down-regulation of miR-30a during chief cell transdifferentiation into SPEM. MiR-30a level was also reduced in the early stage of GC, and its level was maintained in advanced GC. We identified a novel target gene of miR-30a and ITGA2, and our results showed that either ectopic expression of miR-30a or ITGA2 knockdown suppressed GC cell proliferation, migration, and tumorigenesis. Levels of ITGA2 inversely correlated with levels of miR-30a in human intestinal-type GC. We found down-regulation of miR-30a in preneoplastic lesions and its tumor-suppressive functions by targeting ITGA2 in GC. The level of ITGA2, which functions as an oncogene, was up-regulated in human GC. The results of this study suggest that coordination of the miR-30a-ITGA2 axis may serve as an important mechanism in the development of gastric precancerous lesions and intestinal-type GC.

Dysregulation of miRNAs as a signature for diagnosis and prognosis of gastric cancer and their involvement in the mechanism underlying gastric carcinogenesis and progression.

Gastric cancer (GC) is the second main cause of cancer-related mortality worldwide. The poor prognosis and survival of GC are due to diagnosis in an advanced, noncurable stage and with a restricted response to chemotherapy. GC is usually monitored in an advanced stage; therefore, the poor prognosis and lower level of survival rate with a restricted response to chemotherapy can be detected. Valuable and sensible biomarkers are urgently needed to display screen patients with a high risk of GC that can complement endoscopic diagnosis. Such biomarkers will enable the efficient prediction of the therapeutic response and prognosis of GC patients and prefer the establishment of an advantageous treatment method for each and every patient. Noninvasive diagnostic biomarkers may additionally make a contribution to the early identification of GC and enhance medical management. MicroRNAs (miRNAs) are a group of small noncoding RNAs that have displayed a strong association with GC. Accumulating evidence indicates that miRNAs are potential biomarkers with more than one diagnostic function for GC. Actually, miRNAs regulate cell proliferation, apoptosis, migration, invasion, and metastasis via many biological pathways through the repression of target mRNAs. The current review is accordingly to spotlight the multifaceted roles of miRNAs in GC, which would provide indications for future research. Therefore, we review right here the aberrant expression of miRNAs and underlying mechanisms, consequent effects due to miRNAs dysregulation, and accountable target genes in GC. Besides, potential clinical applications are also highlighted.

PRMT5-dependent transcriptional repression of c-Myc target genes promotes gastric cancer progression.

The proto-oncogene c-Myc regulates multiple biological processes mainly through selectively activating gene expression. However, the mechanisms underlying c-Myc-mediated gene repression in the context of cancer remain less clear. This study aimed to clarify the role of PRMT5 in the transcriptional repression of c-Myc target genes in gastric cancer.Methods: Immunohistochemistry was used to evaluate the expression of PRMT5, c-Myc and target genes in gastric cancer patients. PRMT5 and c-Myc interaction was assessed by immunofluorescence, co-immunoprecipitation and GST pull-down assays. Bioinformatics analysis, immunoblotting, real-time PCR, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism.Results: We found that c-Myc directly interacts with protein arginine methyltransferase 5 (PRMT5) to transcriptionally repress the expression of a cohort of genes, including PTEN, CDKN2C (p18INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, to promote gastric cancer cell growth. Specifically, we found that PRMT5 was required to promote gastric cancer cell growth in vitro and in vivo, and for transcriptional repression of this cohort of genes, which was dependent on its methyltransferase activity. Consistently, the promoters of this gene cohort were enriched for both PRMT5-mediated symmetric di-methylation of histone H4 on Arg 3 (H4R3me2s) and c-Myc, and c-Myc depletion also upregulated their expression. H4R3me2s also colocalized with the c-Myc-binding E-box motif (CANNTG) on these genes. We show that PRMT5 directly binds to c-Myc, and this binding is required for transcriptional repression of the target genes. Both c-Myc and PRMT5 expression levels were upregulated in primary human gastric cancer tissues, and their expression levels inversely correlated with clinical outcomes.Conclusions: Taken together, our study reveals a novel mechanism by which PRMT5-dependent transcriptional repression of c-Myc target genes is required for gastric cancer progression, and provides a potential new strategy for therapeutic targeting of gastric cancer.

Methylomics analysis identifies a putative STAT3 target, SPG20, as a noninvasive epigenetic biomarker for early detection of gastric cancer.

Gastric cancer is a leading cause of cancer worldwide. Our previous studies showed that aberrant activation of JAK/STAT3 signaling confer epigenetically silences STAT3 target genes in gastric cancer. To further investigate the clinical significance of this phenomenon, we performed Illumina 850K methylation microarray analysis in AGS gastric cancer cells, and cells depleted of STAT3. Integrative computational analysis identified SPG20 as a putative STAT3 epigenetic target, showing promoter hypomethylation in STAT3-depleted AGS cells. Bisulphite pyrosequencing and qRT-PCR confirmed that SPG20 is epigenetically silenced by promoter hypermethylation in a panel of gastric cancer cell lines including AGS cells, but not in immortalized gastric epithelial GES cells. Expression of SPG20 could be restored by the treatment with a DNMT inhibitor, further suggesting that SPG20 is epigenetically silenced by promoter methylation. Clinically, a progressive increase in SPG20 methylation was observed in tissues samples from gastritis (n = 34), to intestinal metaplasia (IM, n = 33), to gastric cancer (n = 53). Importantly, SPG20 methylation could be detected in cell-free DNA isolated from serum samples of gastritis, IM and gastric cancer patients, having a progressive similar to tissues. Taken together, SPG20, a potential STAT3 target, is frequently methylated in gastric cancer, representing a novel noninvasive biomarker for early detection of this deadly disease.

TFAP2E methylation promotes 5‑fluorouracil resistance via exosomal miR‑106a‑5p and miR‑421 in gastric cancer MGC‑803 cells.

Hypermethylation of transcription factor activating enhancer-binding protein 2e (TFAP2E) has been reported to be associated with chemoresistance to 5-fluorouracil (5-FU) in gastric cancer (GC). In the present study, the molecular mechanism governing this chemoresistance was investigated. Drug-resistant human GC MGC-803/5-FU cells were established and TFAP2E expression and methylation levels were assessed. Autocrine exosomes from GC culture medium were isolated and characterized. MicroRNA (miRNA) microarray analysis was used to determine the miRNA expression profile of GC cell-derived exosomes. Exosomes collected from MGC-803/5-FU cells were co-cultured with control cells, and 5-Aza-2′-deoxycytidine (5Aza) was added into MGC-803/5-FU cells to investigate the relationship between TFAP2E, exosomes and chemosensitivity. In the present study, it was demonstrated that hypermethylation of TFAP2E resulted in its reduced expression and 5-FU chemoresistance in GC cells. miRNAs miR-106a-5p and miR-421 were highly expressed and regulated the chemoresistance induced by TFAP2E methylation. Target gene prediction using miRBase, TargetScan and PicTar revealed that E2F1, MTOR and STAT3 may be TFAP2E target genes in GC. Collectively, our data support an important role of exosomes and exosomal miRNAs in TFAP2E methylation-induced chemoresistance to 5-FU in GC. These results highlight their potential for miRNA-based therapeutics.

Targeting IRF3 as a YAP agonist therapy against gastric cancer.

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP-TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

BackgroundMicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues.MethodsThe study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs.ResultsProfiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients’ plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression.ConclusionsOur study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.

MicroRNA-1 (miR-1) inhibits gastric cancer cell proliferation and migration by targeting MET.

MicroRNAs (miRs) are short endogenous non-coding RNAs that act as posttranscriptional regulatory factors of gene expression. Downregulation of miR-1 has been reported in gastric cancer; however, the mechanisms underlying its functions via target genes in gastric cancer remain largely unknown. The purpose of this study was to investigate the mechanism by which miR-1 inhibits gastric cancer cell proliferation and migration. The effects of miR-1 on gastric cancer cell proliferation and migration were determined by MTT and wound-healing assays. Cell protein expression of the miR-1 target gene MET was analyzed by Western blotting. Finally, MET expression was evaluated by immunohistochemistry in a stomach tumor tissue microarray (TMA). Ectopic expression of miR-1 inhibited proliferation and migration in both AGS and SGC-7901 gastric cancer cell lines. miR-1 directly targets the MET gene and downregulates its expression. MET siRNA also inhibited proliferation and migration in both cell lines. Immunohistochemistry revealed significantly higher MET expression levels in gastric cancer tissues compared with matched adjacent non-cancer tissues. These findings indicate that the miR-1/MET pathway is a potential therapeutic target due to its crucial role in gastric cancer cell proliferation and migration.

The Interaction Between MiR-141 and lncRNA-H19 in Regulating Cell Proliferation and Migration in Gastric Cancer

Background/Aims: Non-coding RNAs including miRNA and lncRNA had been reported to regulate gene expression and were both related to cancer progression. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition (EMT) process and H19 has also been demonstrated to promote malignancy in various cancers. We aimed to determine the correlation between miR-141 and H19 and their roles in gastric cancer in this study. Methods: H19 and miR-141 expression were detected by qRT-PCR. By bioinformatic analysis and luciferase assay we examined the correlation between H19 and miR-141 in vitro. Results: H19 expression was found to be inversely correlated to miR-141 expression in gastric cancer cells and tissues. H19 promotes malignancy including proliferation and invasion whereas miR-141 suppresses malignancy in human cancer cells. MiR-141 binds to H19 in a sequence specific manner, and suppresses H19 expression and functions including proliferation and invasion. MiR-141 could also regulate H19 target genes and miR-141 inhibitor restores H19 siRNA function, while H19 regulates miR-141 target gene ZEB1. Conclusion: These results were the first to demonstrate that H19 and miR-141 could compete with each other and affect their target genes in gastric cancer, which provide important clues for understanding the key roles of lncRNA-miRNA functional network in cancer.

Abstract 2310: Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer

Abstract Gastric cancer is the second leading cause of cancer worldwide. Epigenetic silencing of tumor-suppressors has emerged as an important underlying mechanism in the gastric carcinogenesis. Previous studies showed that infection of H. pylori activates JAK/STAT3 signaling pathway in gastric cancer. However, the role of this aberrant signaling remains unclear. We hypothesized that activation of JAK/STAT signaling leads to epigenetic silencing of STAT3 target genes in gastric cancer. To test this hypothesis, a constitutively activated mouse STAT3 mutant (STAT3c) was transfected into MKN28 gastric cancer cells in which the JAK/STAT signaling pathway is inactive. STAT3c stable transfectant (S16) showing hyperphosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control (C9). Integrative expression microarray coupled with bioinformatic analysis identified putative STAT3 targets, NR4A3 that are down-regulated in S16 cells. In association with up-regulation of DNMT1, NR4A3 exhibited increased promoter methylation in S16 but not C9 cells as demonstrated by bisulphite sequencing and demethylation treatment. Interestingly, NR4A3 was also found to be epigenetically silenced in AGS cells where JAK/STAT signaling is constitutively activated. ChIP-PCR experiment revealed that STAT3 bound to the putative STAT3 binding site in NR4A3 promoter of AGS cells. Depletion of STAT3 by lenti-viral knockdown restored NR4A3 expression in this cell. Interestingly, luciferase reporter assay using the NR4A3 promoter containing putative STAT3 binding site exhibited 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 formation assay (P < 0.001). In clinical specimens, quantitative MSP demonstrated a significant correlation between the degree of NR4A3 methylation and STAT3 nuclear translocation in 72 gastric tumor samples (P < 0.05). Importantly, methylation of NR4A3 was significantly associated with patients with shorter survival (P < 0.05). In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling confers epigenetic silencing of a potential tumor suppressor, NR4A3 in gastric cancer. Methylation of NR4A3 may be able to serve as a prognostic indicator in gastric cancer patients. Citation Format: Michael W. Y. Chan, Li-Han Zeng, Liang-Yu Chang, Claudia Dittner, Jian-Liang Chou, Yao-Ting Huang, Alfred S.L. Cheng, Jiayuh Lin, Kun-Tu Yeh. Epigenetic silencing of a potential tumor suppressor NR4A3 by aberrant JAK/STAT signaling predicts prognosis in gastric cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2310. doi:10.1158/1538-7445.AM2014-2310

Amplification of the miR-181c/d cluster is inversely correlated with PDCD4 expression in gastric cancer

miR-181c/d is dysregulated in gastric cancer (GC). We investigated the amplification and expression of miR-181c/d and its predicted target genes in GC. Amplification of miR-181c/d was quantified by genomic real-time PCR in GC and adjacent normal tissues, as well as the levels of mature miR-181c/d was performed by real-time PCR in the same tissues. The potential target genes of miR-181c/d were predicted using bioinformatics software. Expression of one potential target gene, PDCD4, was measured by semi-quantitative RT-PCR, real-time PCR, and immunohistochemistry. Next, the relationship between miR181c/d expression and PDCD4 expression was analyzed. Results indicated that the amplification and expression of miR-181c/d were significantly higher in GC than in adjacent normal tissues (primary miR-181c/d, P < 0.001; miR-181c, P = 0.0344; miR-181d, P = 0.0153), and there was a strong correlation between mature miR-181c/d and primary miR-181c/d. Thirty-two target genes were predicted, including PDCD4 which is a known tumor suppressor gene. Expression of PDCD4 was significantly down-regulated in GC as compared to adjacent normal tissues and was inversely correlated with miR-181c/d expression in GC (miR-181c and PDCD4: R = −0.496, P = 0.008; miR-181d and PDCD4: R = −0.454, P = 0.003). Therefore, miR-181c/d may play a pivotal role in the pathogenesis of GC by down-regulating PDCD4 expression.

High-level copy number gains of established and potential drug target genes in gastric cancer as a lead for treatment development and selection

The overall survival rate of patients with advanced gastric cancer is poor. Therefore, there is an urgent need for new treatment options for these patients. The identification of drug target genes located on DNA regions exhibiting high-level copy number gains (CNG) may be an effective approach, as has e.g. previously been shown for HER2. The aim of the present study was to identify putative drug targets in patients with gastric cancer by applying this strategy. Genome-wide array comparative genomic hybridization (array CGH) data available from 183 primary gastric cancer samples were analyzed through Ingenuity Pathway Analysis (IPA) to assess whether any established or potential anticancer drug target genes showed high-level CNG, including focal amplifications. A total of 147 high-level gained regions were identified in the gastric cancer samples, harboring 167 genes that had previously been annotated as drug target genes. Thirty (18 %) of these genes showed high-level gains in at least 2 % of the tumors. The identified drug target genes included those for drugs known to be active in advanced (gastric) cancer, targets for targeted therapies in clinical development, as well as targets for drugs currently used for other indications but of potential interest for anticancer treatment. In addition, 12 potential drug target genes were identified, including genes involved in growth factor signaling and cell cycle regulation. The majority of gastric cancers carried one or more high-level CNGs or focal amplifications encompassing putative drug target genes. A number of the associated drugs are currently not being considered for treatment of gastric cancer. Based on these results we hypothesize that DNA copy number profiling may be a useful tool to identify new drug targets and to guide individualized treatment strategies in patients with gastric cancer.

Multiple-to-Multiple Relationships between MicroRNAs and Target Genes in Gastric Cancer

MicroRNAs (miRNAs) act as transcriptional regulators and play pivotal roles in carcinogenesis. According to miRNA target databases, one miRNA may regulate many genes as its targets, while one gene may be targeted by many miRNAs. These findings indicate that relationships between miRNAs and their targets may not be one-to-one. However, many reports have described only a one-to-one, one-to-multiple or multiple-to-one relationship between miRNA and its target gene in human cancers. Thus, it is necessary to determine whether or not a combination of some miRNAs would regulate multiple targets and be involved in carcinogenesis. To find some groups of miRNAs that may synergistically regulate their targets in human gastric cancer (GC), we re-analyzed our previous miRNA expression array data and found that 50 miRNAs were up-regulated on treatment with 5-aza-2'-deoxycytidine in a GC cell line. The “TargetScan” miRNA target database predicted that some of these miRNAs have common target genes. We also referred to the GEO database for expression of these common target genes in human GCs, which might be related to gastric carcinogenesis. In this study, we analyzed two miRNA combinations, miR-224 and -452, and miR-181c and -340. Over-expression of both miRNA combinations dramatically down-regulated their target genes, DPYSL2 and KRAS, and KRAS and MECP2, respectively. These miRNA combinations synergistically decreased cell proliferation upon transfection. Furthermore, we revealed that these miRNAs were down-regulated through promoter hypermethylation in GC cells. Thus, it is likely that the relationships between miRNAs and their targets are not one-to-one but multiple-to-multiple in GCs, and that these complex relationships may be related to gastric carcinogenesis.

Abstract 1147: Expression of ERO1L gene is poor prognostic factor for gastric cancer.

Abstract Gastric cancer is the second cause of cancer-related deaths worldwide. Even though within same stage, gastric cancer patients present diverse clinical manifestations and prognosis. Molecular markers will be important in predicting patients’ outcomes and tailoring personalized treatments according to individual biology. In this study, we analyzed the gene expression profile of human gastric cancer (published on CCR) to identify potential biomarkers. We found that TXN family genes and ERO1L were significantly overexpressed and related to prognosis. We evaluated ERO1L significantly overexpressed in gastric cancer and ERO1L was very highly expressed in hypoxic condition. The other study has identified ERO1L as included in the small group of eight genes predicting poor survival of patients with pulmonary adenocarcinoma. To investigate the function of ERO1L gene in gastric cancer cell line (AGS, MKN1 and NCI-N87), we tested the effect of shRNA inhibition of ERO1L on gastric cancer cells. To determine the biologic role of ERO1L in regulating cancer cell proliferation, stable transfection of shRNA for ERO1L in gastric cancer cells. Our results showed that shERO1L decrease cell proliferation. Furthermore, Knock down expression by shERO1L have effects on regulating gastric cancer cell apoptosis. Next, we tested whether or not the ERO1L gene is involved in progression to metastatic disease in gastric cancer,especially in late tumorigenesis, including migration and invasion. As a results, Knock down expression for ERO1L significantly decreased the migration and invasiveness of gastric cancer cells. In conclusion, our findings show that a prognostic molecular signature that can predict the poor progression of gastric cancer tumors. Furthermore, unequal distribution of expression patterns reflecting activation of ERO1L with different survival rates supports a personalized target therapy in gastric cancer with biomarker gene signature driven patient selection. While further work is needed to elucidate the biological contributions of these markers in. in vivo, the results presented here provide a basis for future investigations of the functional and clinical effect of new target genes in gastric cancer. Citation Format: So-Young Seol, Jae Yun Lim, Sun Och Yoon, Soon Won Hong, Jong Won Kim, Seung Ho Choi, Jae Yong Cho. Expression of ERO1L gene is poor prognostic factor for gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1147. doi:10.1158/1538-7445.AM2013-1147

Abstract 4536: Identification of new prognostic biomarker of gastric cancer by systems analysis

Abstract Gastric cancer is one of the most common and cancer-related deaths worldwide and accounts for an estimated 800,000 deaths annually. Prognosis depends on the stage of disease. Even though within same stage, gastric cancer patients present diverse clinical manifestations and prognosis. To differentiate the malignant potential appropriately, many efforts have been made to select prognostic markers. Molecular markers will be important in predicting patients’ outcomes and personalized treatments according to individual biology. In this study, we analyzed the gene expression profile of human gastric cancer (published on CCR) to identify potential biomarkers which could be used to classify patients according to prognosis and also become new therapeutic targets. We found that TXN family genes and ERO1L were significantly overexpressed and related to prognosis. qRT-PCR and tissue microarray validated TXN overexpression and ERO1L have poor prognosis in gastric cancer. Finally, systems analysis revealed TXN family genes are highly correlated with many oncogene and tumor suppressor functional genes and shown relationship on energy and protein synthesis. Survival analysis based on Hierarchical-clustering of TXN family correlated mRNA expression genes revealed that TXN and TXNIP are significantly influenced the patient survival. We evaluated ERO1L and find out also significantly overexpressed in gastric tumor and suggest TXN family members are actively involved in gastric cancer. In vitro hypoxic experiment ERO1L was very highly expressed in hypoxic condition and this also supports TXN high expressed cancer cells are in hypoxic condition. In conclusion, our findings show that a prognostic molecular signature that can predict the poor progression of gastric cancer tumors. Furthermore, unequal distribution of expression patterns reflecting activation of TXN family and ERO1L with different survival rates supports a personalized target therapy in gastric cancer with biomarker gene signature driven patient selection. While further work is needed to elucidate the biological contributions of these markers, the results presented here provide a basis for future investigations of the functional and clinical effect of new target genes in gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4536. doi:1538-7445.AM2012-4536

Abstract 3941: Amplifications of drug target genes in gastric cancer: Potential clinical implications

Abstract Background: The treatment of advanced gastric cancer remains a challenge for the clinical oncologist. Once distant metastases are present, median survival is around one year with the most active chemotherapeutic regimen. Besides the recent approval of trastuzumab in combination with chemotherapy, thus far no other molecular targeted agents have been approved for the treatment of this disease. There is an urgent need to improve selection of effective treatment for individual patients. A potentially promising approach for selecting active antitumor agents in individual patients could be the evaluation of tumor DNA for amplifications of genes coding for drug targets. Material and Methods: 183 primary gastric adenocarcinomas were analyzed by genome wide array Comparative Genomic Hybridization (arrayCGH) using microarrays consisting of 5000 BAC clones. High level amplifications were defined as a minimum of 2 consecutive clones showing a log2 tumor to normal ratio of &amp;gt; 1. Amplification frequencies for each BAC clone were calculated among the 183 tumors, and genes located within the amplicons were identified by using the RefSeq genes in the UCSC genome browser, version hg16/NCBI34. Drug target genes were selected with Ingenuity Pathway Analysis (IPA) and the Pharmacogenomics Knowledge Base (PharmGKB). Genes amplified in at least 4 patients (2%) were classified according to the mechanism of action of the drug interacting with this specific gene. Results: In 183 gastric cancers a mean number of 2.0 amplifications per tumor (range 0-21) was found. 167 genes out of more than 6000 at these loci were annotated as drug target genes by IPA, with 30 genes having an amplification frequency of at least 4 (2%). Most frequently amplified regions were located on chromosome 6, 8, 11, 16, 17 and 20. Genes for molecular targeting agents included VEGFA (6.5%), ERBB2 (6%), HSP90AB1 (5.5%) and EGFR (3.8%). Interestingly, also genes for non-anticancer agents, including HRH3 (10.4 %) and RARA (4.9%), and potential therapeutic targets such as TNFRSF4B (7.1%) were found. Conclusion: ArrayCGH analysis of a large series of gastric tumors revealed many non-randomly amplified gene loci, including substrates for targeted agents with potential antitumor activity in advanced gastric cancer. Moreover, targets of agents without established anticancer activity were identified as well. Our results provide a molecular rationale for the investigation of new treatment strategies in advanced gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3941. doi:10.1158/1538-7445.AM2011-3941

Abstract 1232: Aberrant JAK/STAT signaling confers epigenetic silencing of STAT3 targets in gastric cancer by integrated microarray and bioinformatic analysis

Abstract Gastric cancer is the 2nd leading cause of cancer worldwide. One of the major risk factors of gastric cancer is infection of H. pylori. Patients infected with CagA-positive H. pylori would have increased risk of gastric cancer. Previous studies including ours have demonstrated that infection of CagA-positive H. pylori induced activation of JAK/STAT3 signaling pathway. However, the role of aberrant activation of this signaling pathway in gastric cancer is not clear understood. We hypothesized that aberrant activation of JAK/STAT signaling may lead to epigenetic silencing of STAT3 target genes in gastric cancer. In this study, we transfected a constitutively activated mouse STAT3 mutant (STAT3c) into MKN28 gastric cancer cell line in which the JAK/STAT signaling pathway is not active. STAT3c stable transfectant MKN28/STATc-16 showing hyper-phosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control MKN28/C-9. In order to identify STAT3 target genes that are epigenetically down-regulated in MKN28/STATc-16 cells, expression microarray using Agilent 44K oligonucleotide microarray coupled bioinformatic analysis were performed. Our result showed that there are 2945 genes showing at least 1.5 fold down-regulation in MKN28/STATc-16 cells as compared with vector control. By whole genome bioinformatic analysis, 397 genes with STAT3 binding sites within 5kb of a CpG island were discovered. Integrated analysis revealed that there are 18 downregulated genes with a STAT3 binding site around the promoter CpG island. Further characterization of two STAT3 target, NR4A3 and LOXL4 demonstrated that promoter hypermethylation of these 2 genes were observed in MKN28/STATc-16 cells but not the vector control. Demethylation treatment using 5azaDC also restored expression of these 2 genes in MKN28/STATc-16 cells. These phenomena may be partially related to the up-regulation of DNMT1 in MKN28/STATc-16 cells. In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling may lead to epigenetic silencing of its target gene in gastric cancer. The role of STAT3 in mediating epigenetic silencing of NR4A3 and LOXL4 and its clinical significance in gastric cancer patients is currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1232.

Abstract 200: Methylation of GATA4 and GATA5 transcription factor genes in gastric cancers

Abstract GATA factors comprise a small family of transcription regulatory proteins containing two conserved zinc finger DNA binding domains for the consensus sequence (WGATAR). Among the six GATA factors, GATA-1/2/3 factors are prominently expressed in the hematopoietic cell lineage, while GATA-4/5/6 factors show a partly overlapping expression pattern in the heart and endoderm-derived organs. We previously reported that GATA4 and GATA5 were silenced by their promoter hypermethylation in gastrointestinal, lung and ovarian cancers. However, it remains unknown the mechanism underlying loss of GATA transcription factors in gastric carcinogenesis. To clarify the role of GATA, we examined GATA4/5 methylation and their downstream target genes in gastric cancer (GC). By using methylation specific PCR, the methylation frequency of GATA5 (30, 39%) was significantly higher than that of GATA4 (10, 13%) in 77 GC tissues (P&amp;lt;0.01). However, methylation of GATA6 was not observed in GCs examined. We transiently over-expressed GATA4 or GATA5 in GC cell lines lacking their expression by aberrant methylation. Both GATA4 and GATA5 suppressed colony formation and cell proliferation in MKN74 cells. After GATA4/5 over-expression in MKN74 and KATO-III cells, enhanced expression of numerous genes including ones indicating tumor-suppressive functions, such as TFF1, disabled-2 and IQGAP2, was observed by microarray and RT-PCR analyses. The target genes of GATA4 and GATA5 were closely similar in MKN74 cells. We also preformed siRNA transfection of GATA4 and GATA5 in AGS and HSC43 cells which basally expressed them. Reduced expression of above predicted GATA target genes was detected in these two cell lines after siRNA transfection. These data suggest that loss of GATA4 and GATA5 expression may contribute to gastric carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 200.