Subset Of Gastric Cancers Research Articles

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.

Abstract B43: A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy.

Abstract Gastric carcinoma (GC) is a common cancer of high mortality and particularly prevalent in East Asia. There are only a few treatment options: surgery, chemotherapy, as well as Herceptin® for a small subset of patients with erbb2 amplification. Cetuximab has been approved for CRC (colorectal) and SCCHN, but not yet for gastric carcinoma. Cetuximab, combined with capecitabine/cisplatin, has recently been tested in Phase III clinical trial (EXPAND) in GC patients (1), but failed to demonstrate benefit over the existing chemotherapy (capecitabine/cisplatin). One of the reasons for the failure is only small subset of patients responding to cetuximab, which constitutes little effects on large trial populations, while the identity of the responders yet to be revealed. One of the keys to a successful search for effective treatment is the development of experimental model that truly mimics patient conditions. Patient derived xenograft (PDX or HuPrime®) is believed to best mimic human cancers (2). Recently, we have established a cohort of ∼70 GC xenograft (PDX) models from Asian and Caucasian patients. In the present study, we conducted a mouse clinical trial (MCT, or PDX trial) to test cetuximab in a cohort of GC-PDX, aiming at identifying those responsive to cetuximab. Interestingly, our trial result identified 4 of 20 (20%) GC-PDX responded to cetuximab. Genome-wide profiling and additional investigations revealed that high EGFR mRNA expression and immunohistochemistry score (3+) are associated with tumor growth inhibition. Furthermore, EGFR amplification were observed in 4/4 (50%) responders by DNAchip analysis or 2/4 confirmed with FISH analysis, with average copy number 5.8 and >15 respectively. Our data suggest that a GC subtype with EGFR amplification and overexpression benefit from cetuximab treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B43. Citation Format: Lianhai Zhang, Jie Yang, Yiyou Chen, Jie Cai, Xiaoming Song, Xuesong Huang, Jianyun Deng, Dawei Chen, Mengmeng Yang, Shuangxi Li, Aiwen Wu, Ziyu Li, Zhongwu Li, Yiqian Liu, Jean-Pierre Wery, Henry Li, Jiafu Ji. A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B43.

A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy

A preclinical trial identified 4 of 20 (20%) gastric cancer (GC) patient-derived xenografts responded to cetuximab. Genome-wide profiling and additional investigations revealed that high EGFR mRNA expression and immunohistochemistry score (3+) are associated with tumor growth inhibition. Furthermore, EGFR amplification were observed in 2/4 (50%) responders with average copy number 5.8 and >15 respectively. Our data suggest that a GC subtype with EGFR amplification and overexpression benefit from cetuximab treatment.

Tu1612 Reprimo, a p53 Dependent G2 Arrest Mediator, Is Downregulated in the Signet-Ring Cell Type, a Particularly Aggressive Subset of Gastric Cancer

Tu1612 Reprimo, a p53 Dependent G2 Arrest Mediator, Is Downregulated in the Signet-Ring Cell Type, a Particularly Aggressive Subset of Gastric Cancer

Abstract 2090: Targeting HER family signaling in gastric cancer: The selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, is efficacious in primary models of HER family driven disease.

Abstract Despite an overall decline, gastric cancer incidence remains high in Asian countries including China, Japan and South Korea. Five-year survival rates continue to improve in early stage disease through an aggressive combination of surgery and chemo/radiotherapy, primarily using fluoropyrimidine and platinum agent combinations. In advanced disease, the TOGA trial provided clinical validation of a role for HER2 in driving tumor progression in a subset of gastric cancer (GC) patients. However, recent clinical studies with EGFR targeting antibodies in GC have failed to show promise (EXPAND and REAL3 trials). Nonetheless, the dismal prognosis associated with late stage disease and the frequent implication of dysregulated HER family signaling in GC necessitate further investigation. AZD8931 is an orally bioavailable, highly selective and potent small molecule inhibitor of EGFR, HER2 and HER3 signaling(1). To evaluate the potential utility of this agent in clinical GC, we examined HER family gene amplification and expression in GC tissue samples and sought to correlate these profiles with response to AZD8931 in GC tumor xenograft models. High Her2 expression (IHC3+ or IHC2+/FISH+ve) was confirmed in 17.4% of tumours (20/115). Importantly, high level EGFR (44.4%, 48/108) and HER3 (89.3%, 92/103) protein expression was also detected in our cohort of Chinese GC samples. Gastric cancer cell panel screening (n=25 lines) identified a number of AZD8931-sensitive cell lines with a range of HER family expression profiles (GI50 range 0.02 to 0.35μM). In the NUGC3 xenograft model (high EGFR expression), twice-daily oral dosing of AZD8931 elicited potent, dose-responsive anti-tumour activity at well-tolerated doses (98% TGI at 6mg/kg/bid, 108% TGI at 12.5mg/kg/bid, all P<0.0001) and anti-tumour efficacy correlated well with pharmacodynamic modulation of phospho-HER signaling. To investigate the translational relevance of these data, primary GC models with varying HER family protein expression profiles were identified and established. Within these models, AZD8931 anti-tumor efficacy correlated broadly with strong HER pathway activation (0 to 83% TGI at 12.5mg/kg/bid). We further evaluated the activity of AZD8931 in combination with cisplatin/5-FU, 1st line standard of care chemotherapies commonly used in the treatment of gastric cancer. Using a primary GC xenograft model, AZD8931 demonstrated additive benefit when used at well tolerated doses (minimal body weight loss). These data support the potential clinical utility of AZD8931 for the therapeutic treatment of gastric cancers exhibiting strong HER pathway signaling. 1 Hickinson et al. Clin.Cancer Res 2010; Feb 15. Citation Format: Paul R. Gavine, Liang Xie, Lin Zhang, Jing Lv, Lu Han, XiaoLu Yin, Yuan Jie Liu, Xinying Su, Shuqiong Fan, Teresa Klinowska. Targeting HER family signaling in gastric cancer: The selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, is efficacious in primary models of HER family driven disease. [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 2090. doi:10.1158/1538-7445.AM2013-2090

Identification of ROS1 rearrangement in gastric adenocarcinoma

Recently, chromosomal rearrangements involving receptor tyrosine kinases (RTKs) have been described in common epithelial malignancies, including nonsmall cell lung cancer (NSCLC), colorectal cancer, and breast cancer. One of these RTKs, c-ros oncogene 1, receptor tyrosine kinase (ROS1), has been identified as a driver mutation in NSCLC, because its inhibition by crizotinib, an anaplastic lymphoma receptor tyrosine kinase (ALK)/met proto-oncogene hepatocyte growth factor receptor (MET)/ROS1 inhibitor, led to significant tumor shrinkage in ROS1-rearranged NSCLC. Currently, only human epidermal growth factor 2 (HER2)-targeted therapy in combination with chemotherapy has been successful in significantly prolonging the survival of patients with advanced gastric cancer (GC). There is a need for the discovery of additional novel targets in GC. Anti-ROS1 immunohistochemistry (IHC) was used to screen 495 GC samples and was followed by simultaneous ROS1 break-apart fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses in IHC-positive samples. Fusion partners in ROS1-rearranged GC were determined by RT-PCR. In all 495 samples, HER2 amplification was identified with FISH, and MET expression was identified by IHC. Twenty-three tumor samples were ROS1 IHC-positive. Three of 23 patients were ROS1 FISH positive, HER2 FISH negative, and negative for MET overexpression; and 2 of those 3 patients harbored a solute carrier family 34 (sodium phosphate), member 2 (SLC34A2)-ROS1 fusion transcripts. No fusion partner was identified in the third patient. Both patients who had SLC34A2-ROS1 transcripts had poorly differentiated histology with recurrence and death within 2 years of curative surgery. ROS1 IHC-positive status was not identified as an independent prognostic factor for overall survival. In this study, an SLC34A2-ROS1 rearrangement was identified in GC, and the results provide a rationale for investigating the clinical efficacy of ROS1 inhibitors in this unique molecular subset of GC. Society.

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer

BackgroundGenomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer.Methods and ResultsA genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide.ConclusionThese results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

Abstract 3682: Overexpression and cell proliferation-regulating function of CRKL protein in gastric cancer

Abstract Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. To identify previously undiscovered amplified genes in gastric cancer, a genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers. Twenty-two genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, which has not been previously characterized in gastric cancer and the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. A relatively high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization, and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. These results suggested that CRKL protein is overexpressed in a subset of gastric cancers, that it regulates gastric cell proliferation, and that it has the potential to be a molecular therapy target for 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 3682. doi:1538-7445.AM2012-3682

Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodeling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

High CpG island methylator phenotype is associated with lymph node metastasis and prognosis in gastric cancer.

Several studies have found that the promoter CpG island is frequently methylated in gastric cancer. The CpG island methylator phenotype (CIMP) defines concordant methylation of multiple promoter CpG island loci in a subset of gastric cancer. However, the relationship between CIMP and lymph node metastasis in gastric cancer is unknown. Our study aimed to characterize the role of CIMP in lymph node metastasis. Clinical specimens from 120 patients were analyzed and PCR was used to detect the methylation status of five genes (ALX4, TMEFF2, CHCHD10, IGFBP3, and NPR1). We measured the level of mRNA for the five genes by real-time RT-PCR. Microsatellite instability and Helicobacter pylori infection status were assayed by capillary electrophoresis and real-time PCR, respectively. DNA methylation in the five genes was correlated with low expression of the respective mRNA. With CIMP as the dependent variable, CIMP-high gastric cancer tended to show more distant lymph node metastasis, higher pathologic tumor classification, more pathologic metastasis, and higher pathologic TNM status. Microsatellite instability and H. pylori status were not significant predictors of prognosis. CIMP-high gastric cancer showed significantly worse survival compared with that of CIMP-low/CIMP-negative gastric cancer (P < 0.001). Our results show that there is an association between CIMP status and lymph node metastasis in gastric cancer and CIMP-high was an independent prognostic factor.

A phase I study of dovitinib in combination with capecitabine and oxaliplatin in upfront treatment of advanced colorectal and gastric cancer with a dose expansion cohort in advanced gastric cancer.

TPS174 Background: Dovitinib (TKI258) is an investigational multi-targeting tyrosine kinase inhibitor with potent inhibitory activity against several class III,IV,V RTKs relevant in cancer, including VEGF receptors-1,2,3, bFGF receptors-1,2,3, PDGFR and c-KIT. The addition of bevacizumab to chemotherapy is presently employed in management of colorectal cancer but did not improve survival in gastric cancer (AVAGAST). FGF pathway activation has been related to aggressive gastric and colorectal cancer, resistance to anti-VEGF therapy may be mediated in part by FGF upregulation. FGFR2 amplification is reported in subsets of gastric cancer (Hara et al. Lab Invest (1998) 78(9):1143-53). FGFR2 is required for cell proliferation and survival in FGFR2 amplified gastric cancer cell lines (Kunli et al. Cancer Rsch (2008) 68; 2340), which are sensitive to FGFR2 inhibitors, including Dovitinib. Methods: This trial is a single arm open label phase I trial in patients with treatment naïve advanced gastric or colorectal cancer. We will employ the standard “3+3” phase I dose escalation design to determine the recommended phase II dose of dovitinib in combination with standard doses of capecitabine (1000mg/m2 bid d1-d14 q21) and oxaliplatin (130mg/m2 q21). 4 dose levels of dovitinib (5 days on, 2 days off: 200mg, 300mg,400mg,500mg) and a dose level minus 1 have been defined. In a dose expansion cohort, 12 patients with advanced gastric or gastroesophageal adenocarcinoma will be treated with dovitinib, capecitabine and oxaliplatin at the recommended phase II dose (RP2D). Our secondary objectives are to describe the toxic effects and pharmacokinetics of dovitinib when administered in combination with capecitabine/oxaliplatin and identify any activity of the TKI258/XELOX treatment combination in gastric and colorectal cancer. Exploratory pharmacodynamic investigations as well as molecular studies to deeply characterize patient tumors will be performed.

Abstract 1643: AZD4547, a potent and selective inhibitor of FGF-receptor tyrosine kinases 1, 2 and 3, inhibits the growth of FGF-receptor 2 driven gastric cancer models in vitro and in vivo

Abstract Gastric cancer is the second most lethal cancer worldwide. We and others have shown that the FGFR2 gene is amplified in a subset of gastric cancers. To validate FGFR2 as a potential therapeutic target for gastric cancer, we employed an inducible shRNA system to specifically block expression of individual FGFRs, both in vitro and in vivo. When FGFR2 was specifically down regulated in an FGFR2-amplified gastric cancer cell line Snu-16, the activation of pFGFR2 and its downstream signaling effectors were blocked and there was significant inhibition of the cell growth and survival that further translated into tumor growth regression in vivo, demonstrating that in this model tumor growth/survival is dependent on the FGFR2 pathway. To further confirm this, AZD4547, a selective inhibitor of FGFR 1, 2 and 3 receptor tyrosine kinases was tested in a panel of gastric cancer cell lines for its anti-proliferative effect. In this panel the cell-lines that were most sensitive to AZD4547 were the Snu-16 and KATOIII, both of which are gene amplified for FGFR2. Exposure of the Snu-16 cells to AZD4547 resulted in cell-death and once daily oral dosing of AZD4547 resulted in tumor growth inhibition of the Snu-16 xenografts in a dose-dependent fashion, with significant tumor regression observed when AZD4547 was dosed once daily at 12.5 mg/kg. The anti-tumor efficacy of AZD4547 correlated with inhibition of pFGFR2 and modulation of other downstream markers of FGFR pathway activation, including pErk, pS6 protein and pPLCγ and efficacious doses of AZD4547 induced elevations of levels of plasma FGF23, a known marker of FGFR inhibition. In addition to the single-agent efficacy, combining of AZD4547 with cytotoxic agents, currently used for the treatment of gastric cancer patients, revealed enhanced growth inhibition of Snu-16 xenografts. Taken together, these data support clinical testing of AZD4547 in gastric cancer patients harboring FGFR2 gene amplification. 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 1643. doi:10.1158/1538-7445.AM2011-1643

Abstract 2129: HB-EGF expression in gastric cancer promotes chemotherapy resistance and represents a promising target for molecular therapy and a novel biomarker of 5-FU resistance

Abstract The incidence of gastric cancer (GC) has been on the decline in the U.S. over the last 40 years, but remains high in Asia and Latin America. The primary treatment for early-stage GC is surgery. Many patients will also benefit from adjuvant 5-fluorouracil (5-FU) or platin-based chemotherapy. When it presents at an advanced stage, GC uniformly carries a poor prognosis, due to chemotherapy resistance. To improve the response of GC to chemotherapy, molecular-targeted therapies are needed. One of the EGFR family ligands called heparin-binding epidermal growth factor (HB-EGF) was recently found to be up-regulated in several epithelial-based malignancies, including GC. Our objective in this study was to evaluate the role of HB-EGF in GC chemotherapy resistance. We hypothesize that constitutive expression of HB-EGF is associated with chemotherapy resistance. Furthermore we propose that inhibition of HB-EGF enhances drug sensitivity and augments the apoptotic response to chemotherapy in GC. To test our hypothesis, we isolated RNA and protein from a panel of 6 GC cell lines and measured the basal HB-EGF expression compared to that of normal gastric epithelia. We similarly measured HB-EGF expression in response to 5-FU or cisplatin treatment, and then determined the response when cells were co-treated with HB-EGF or an inhibitor of HB-EGF (CRM197). We found that HB-EGF expression at both the protein and RNA levels was highly variable across the panel of GC cell lines. In addition, HB-EGF expression increased in response to drug treatment at 24 and 48 hours post-treatment. A positive correlation was observed between HB-EGF mRNA levels and chemoresistance to 5-FU. GC cell lines that are high-expressers of HB-EGF showed decreased cell growth when treated with CRM197, whereas low-expressers increased when treated with recombinant HB-EGF. The combination of CRM197 with anticancer agents increased apoptosis in high-expressers, but made no significant difference in low-expressers. Likewise, HB-EGF gene silencing using a lentiviral short-hairpin RNA significantly decreased high-expressers survival and proliferation. Similar to our findings using GC cell lines, we found that human gastric cancer tumor samples showed highly variable HB-EGF expression compared to their matching normal gastric epithelia. Our findings suggest that HB-EGF plays an important role in the proliferation and chemotherapy resistance of a subset of gastric cancers. HB-EGF may represent a promising molecular target in those select patients, in whom the gastric cancer expresses high levels of HB-EGF. Furthermore, HB-EGF expression may represent a novel biomarker for 5-FU chemoresistance in patients with 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 2129. doi:10.1158/1538-7445.AM2011-2129

Frequent down‐regulation of hABH2 in gastric cancer and its involvement in growth of cancer cells

Methyl or 1, N(6) -ethenoadenine base lesions are frequent and highly-mutagenic or -carcinogenic events in mammalian DNA. Human AlkB homologue-2 (hABH2), a homologue of the Escherichia coli AlkB protein, has been found to be the principal dioxygenase for the repair of these lesions. Mounting evidence indicates that impaired DNA repair contributes to gastric cancer induction and progression. Whether hABH2 is involved in this malignancy is unknown. The present study was aimed to investigate the expression profile of hABH2 in gastric cancer and the effect of hABH2 on cancer cell growth. The expression of hABH2 in 35 pair-matched gastric neoplastic and adjacent non-neoplastic tissues, and in five gastric cancer cell lines, was examined by real-time polymerase chain reaction (PCR), immunohistochemistry, or Western blot. The cell growth was determined using cell-counting kit-8 assay. The apoptosis or cell-cycle analysis was determined using flow cytometry. The hABH2 expression was downregulated in 68% (24/35) of primary gastric cancers, as determined by real-time PCR; the hABH2 expression was also substantially decreased in gastric cancer cell lines. Immunohistochemical or Western blot analysis further confirmed the downregulation of hABH2 expression in gastric cancers. The overexpression of hABH2 significantly inhibited the proliferation of gastric cancer cells, and induced G(1) arrest of the cell cycle, while hABH2 knockdown promoted cell growth and cell-cycle progression of gastric cancer cells. These results suggest that hABH2 is downregulated in a subset of gastric cancers, and might be involved in the molecular mechanism of gastric cancer through inhibiting the proliferation of gastric cancer cells.

Abstract 284: FGFR2 is required for growth and survival in a rare subset of FGFR2 amplified colorectal cancer

Abstract FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (&amp;lt; 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. 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 284.

Abstract 2802: Altered expression and the missense polymorphism of the human base excision repair gene NTH1 in gastric cancer

Abstract Reactive oxygen species generated in living cells during normal cellular metabolism and in response to exogenous inducers cause the formation of oxidatively damaged bases in DNA. These oxidized base lesions are primarily removed by the base excision repair (BER) pathway. A BER enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol, from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against thymine glycol by empty-vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced mRNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. Next, we found two novel genetic polymorphisms, i.e., c. −163C&amp;gt;G and c. −241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. Next, all the coding exons of the NTH1 gene and their boundary regions in DNAs of 23 Japanese patients with gastric cancer was searched for genetic polymorphisms by direct sequencing. A novel variant, c. −31A&amp;gt;G, and a single nucleotide polymorphism, c.98G&amp;gt;A (Arg33Lys, rs2302172) were detected at an allele frequency of 2.2%, and 4.3%, respectively. However, the c. −31A&amp;gt;G polymorphism did not affect reduction of promoter activity. We are going to reveal functional characterization of c.98G&amp;gt;A and to examine excisional repair activity against new substrates, such as oligonucleotide containing etheno adducts. Our data suggested that downregulation of NTH1 expression, abnormal localization of NTH1 and the missense polymorphism may be involved in the pathogenesis of a subset of gastric cancers. 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 2802.

Expression profile of REG family proteins REG Iα and REG IV in advanced gastric cancer: comparison with mucin phenotype and prognostic markers

Regenerating gene family members 1 (REG Iα) and 4 (REG IV) are overexpressed in a subset of gastric cancers. However, comparative characterization of the expression of these family proteins has remained unclear. Therefore, we aimed to elucidate not only the association between REG protein expression and mucin phenotype but also their significance as a prognostic marker for patients with gastric cancer. The expression of REG Iα, REG IV, CDX2, MUC2, and MUC5AC in gastric cancer tissues was examined by immunohistochemistry. The relationship between REG protein expression and clinicopathological parameters or mucin phenotype was then analyzed. REG Iα and REG IV expression was positive in 33 (52%) and 31 (49%) of 63 gastric cancers examined, respectively. REG Iα expression was significantly related to venous invasion and tumor stage, whereas REG IV expression showed no relationship to clinicopathological features. With regard to mucin phenotype, REG IV expression was significantly correlated with MUC2 and CDX2 expression, suggesting an association with the intestinal mucin phenotype of gastric cancer. On the other hand, REG Iα expression had no correlation with MUC2, CDX2, or MUC5AC in gastric cancer tissues. Expression of REG Iα but not REG IV was an independent predictor of poor outcome in patients with gastric cancer. In addition, patients with gastric cancer negative for both REG Iα and REG IV expression had a significantly better outcome than patients positive for either REG Iα or REG IV. Profiling of REG protein expression is useful to for prognostication of patients with gastric cancer.

Methylation of PTCH1a gene in a subset of gastric cancers

To establish if PTCH1a transcriptional regulation region (TRR) is methylated in gastric cancer and its influence in gastric tumorigenesis. The CpG islands in PTCH1a TRR were analyzed by Methyl Primer Express v1.0 software. The region from -643 to -355 bp (the transcription initiation site of PTCH1a was designated as 0) that contained 19 CpG sites was chosen for bisulfite-sequencing PCR (BSP) and methylation-specific PCR (MSP) detection. The gastric cancer cell line AGS was treated with 5-aza-2'-deoxycytidine (5-Aza-dC; 1 micromol/L) for 3 d. Alterations in PTCH1a TRR methylation in treated AGS cells was measured through BSP clone sequences, and their PTCH1 expression was measured by quantitative RT-PCR. The cell cycle and apoptosis were observed with flow cytometry through propidium iodide (PI) staining or annexin V/PI double staining. The prevalence of PTCH1a TRR methylation was investigated in 170 gastric cancer tissue samples and the adjacent normal tissues by MSP. The correlation of PTCH1a TRR methylation with PTCH1 expression or with patients' clinical features was analyzed. Methylation of PTCH1a TRR was observed in AGS cells and a subset of gastric cancer tissues (32%, 55/170), while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1a TRR was correlated negatively with PTCH1 expression (Spearman's r = -0.380, P = 0.000). However, methylation of PTCH1a TRR was not related to the gastric cancer patients' clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCH1a TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression (5.3 +/- 2.5 times; n = 3) and apoptosis rate (3.0 +/- 0.26 times; P < 0.05; n = 3). Methylation of PTCH1a TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in gastric tumorigenesis and a new treatment target.

FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival

We have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification.

Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2′-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein–Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.