Prognostic significance of glutathione peroxidase 1 (GPX1) down-regulation and correlation with aberrant promoter methylation in human gastric cancer.

BACKGROUND: This study aimed at examining the association of gene silencing and promoter methylation of glutathione peroxidase 1 (GPX1) and glutathione peroxidase 3 (GPX3) in gastric cancer cells and determined the clinical significance of GPX1 and GPX3 expression loss in gastric cancer tissue. MATERIALS AND METHODS: Analysis of mRNA expression was carried out by reverse transcription-polymerase chain reaction (RT-PCR). Methylation of the GPX1 promoter region was analyzed by bisulfite sequencing, and that of the GPX3 promoter region was analyzed by methylation-specific PCR (MSP). Tissue microarray-based immunohistochemistry of GPX1 and GPX3 in 1,163 resected gastric cancer specimens was performed to assess the associations with clinicopathological parameters. RESULTS: Reduced GPX1 and GPX3 mRNA expression was associated with promoter methylation in gastric cancer cell lines. A correlation between DNA promoter methylation and loss of GPX1 expression was noted in 16 gastric cancer tissue samples (p=0.005). Loss of GPX1 and GPX3 proteins was found in 24.4% and 30.8% of gastric cancer tissues. Loss of GPX1 expression was significantly associated with advanced gastric cancer (p=0.039) and lymphatic invasion (p=0.010); loss of GPX3 expression was associated with advanced gastric cancer (p<0.001) and lymph node metastasis (p<0.001). Kaplan-Meier analysis showed that low expression of GPX1 was associated with poor cancer-specific survival (p=0.010). CONCLUSION: Data from this study implicate aberrant hypermethylation of promoter regions of GPX1 and GPX3 as a mechanism for down-regulation of GPX1 and GPX3 mRNA expression in gastric cancer cells. Loss of GPX1 expression was associated with aggressiveness and poor survival in patients with gastric cancer.

Downregulation of methylthioadenosin phosphorylase by homozygous deletion in gastric carcinoma.

The methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 telomeric to the CDKN2A tumor suppressor gene. Loss of MTAP gene is frequently associated with CDKN2A homozygous deletion. Although the homozygous deletion of MTAP has been reported in various human cancers, its function in gastric carcinogenesis is unknown. Here, we determined the status of the MTAP gene by using a combination of array-based comparative genomic hybridization and oligonucleotide microarray. It was found that MTAP was deleted and downregulated in 2 of 10 gastric cancer cell lines. Of the 494 primary gastric carcinomas examined, MTAP expression at the protein level was reduced in 59 (11.9%). Furthermore, a lack of MTAP expression was found to be associated with poor survival (P = 0.038). The genomic loss of MTAP and CDKN2A in gastric carcinomas was investigated by quantitative real-time PCR. Among 20 gastric carcinomas, two cases showed deletion of both MTAP and CDKN2A, and three samples showed homozygous deletion of MTAP, but not of CDKN2A. An analysis of gastric carcinomas revealed that reduced MTAP expression correlated significantly with a genomic deletion. Furthermore, functional assays by transfecting the siRNA or the expressional cDNA into gastric cancer cell lines demonstrated that MTAP regulates cell growth and invasion. The present study suggests that MTAP plays an important role in the regulation of gastric carcinogenesis and, in particular, that MTAP loss is implicated in some way with tumor growth via the modulation of cellular properties, which, in turn, suggests that MTAP has therapeutic applications.

Reduced expression and homozygous deletion of annexin A10 in gastric carcinoma.

Annexins (ANXs) are a family of calcium and phospholipid binding proteins that have been implicated in diverse important biological and physiological process. ANX A10 (ANXA10) is a member of this family, though little is known about its functions. In the present study, array based comparative genomic hybridization (CGH) was used to screen DNA copy number change in gastric cancer cell lines and the results obtained were compared with oligonucleotide microarray data. DNA loss of the ANXA10 locus in chromosome 4q33 was found in several gastric cancer cell lines by array based CGH and these cell lines showed decreased ANXA10 expression by oligonucleotide microarray analysis. Functional analysis using siRNA and full-length cDNA transfection in gastric cancer cell lines demonstrated that ANXA10 regulates gastric cancer cell proliferation. Of the 585 primary gastric carcinoma tissues examined, ANXA10 expression at the protein level was found to be reduced in 289 (49.4%) cases. Quantitative real-time PCR analysis validated loss of DNA at the ANXA10 locus in gastric carcinomas with reduced ANXA10 expression. By univariate survival analysis, lack of ANXA10 expression was associated with poor survival (p = 0.016). These results suggest that ANXA10 inactivation by chromosomal deletion may play a role during gastric cancer progression.

Identification of genes epigenetically silenced by CpG methylation in human gastric carcinoma.

To identify novel methylation-silenced genes in gastric cancer, we carried out a genome-wide search for genes that are up-regulated after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5Aza-dC). When three gastric cancer cell lines (SNU-1,-601, and -719) were treated with 5Aza-dC, 143 genes were found to be upregulated by twofold or more using oligonucleotide microarrays. Six of these genes, i.e. TFPI2, GPX3, GPX1, IGFBP6, IRF7 and DMRT1, showed promoter hypermethylation in one or more gastric cancer cell lines, but were unmethylated in normal gastric mucosa by bisulphite sequencing and methylation-specific PCR analysis. The following percentages of these genes were found to be aberrantly methylated in gastric cancer samples; TFPI2 (80.9%), GPX3 (30.1%), DMRT1 (46.9%), GPX1 (16.7%), IGFBP6 (22.6%) and IRF7 (32.1%). Interestingly, the survival of patients possessing methylated alleles of TFPI2 (123/152, 80.9%) was poorer than that of patients with unmethylated alleles (p=0.023). Multivariate analysis confirmed that TFPI2 methylation is a significant and independent prognostic factor in gastric carcinoma. Furthermore, altered TFPI2 expression, as demonstrated by immunohistochemistry in 566 consecutive gastric cancer tissues, was found to be significantly associated with sex (p=0.003), WHO classification (p<0.001), and a mixed subtype by Lauren's classification (p<0.001). Thus, the present study identified several novel genes, which were methylated in gastric cancer and among them, methylation of TFPI2 was an unfavourable prognostic marker.

Expression of type 2 hexokinase and mitochondria-related genes in gastric carcinoma tissues and cell lines.

BACKGROUND: The constant overexpression of glycolysis and active mitochondrial function are critical for productive energy required for the immortal proliferation of cancer cells. The genes related to glycolysis and mitochondrial respiration might have some function during stomach carcinogenesis. MATERIALS AND METHODS: The expression of hexokinase 2 (HK2), Bcl-2 and several mitochondria-related gene products were investigated by immunohistochemistry in 257 consecutive human gastric carcinomas, and the results were compared with the clinicopathological characteristics. In addition, transcriptional change of HK2 and Bcl-2 was investigated in the hypoxic state or with mitochondrial inhibitors. RESULTS: In immunohistochemical analysis, HK2 was overexpressed in 43 out of 257 stomach cancer patients. Bcl-2 was not expressed in cases with HK2 positive cancer tissues except for one case, while the voltage-dependent anion channel, complex II and pyruvate dehydrogenase, located in mitochondria, were expressed in all cases. The patients with HK2 expression showed a worse prognosis compared to the HK2 negative cases, and patients who were negative in Bcl-2 and positive in HK2 represented the lowest survival rate. HK2 and Bcl-2 responded to hypoxia, but not to mitochondrial dysfunction while the cellular growth was severely repressed by mitochondrial inhibitors, indicating that transcriptional regulation of HK2 and Bcl-2 proceeds upstream of dysfunctional mitochondria. CONCLUSION: HK2 was overexpressed in 16.7% of gastric carcinomas, and reciprocal expression pattern with Bcl-2. The HK2 positive cases showed a worse prognosis and aggressive character.

Loss of promyelocytic leukemia protein in human gastric cancers.

To clarify the clinical implications of promyelocytic leukemia (PML) expression in gastric carcinomas, the expression of PML was analyzed in large series of gastric carcinoma by immunohistochemistry, western blotting and reverse transcription-PCR. PML protein expression was reduced or abolished in gastric carcinomas (31.7 and 10.6%, respectively) by immunohistochemistry. PML protein loss was associated with more lymphatic invasion, higher pTNM stage, and worse patient survival. Only one gastric carcinoma cell line showed loss of PML, and the PML protein re-appeared after the treatment of proteasome inhibitor in this cell line. We conclude that PML protein loss occurs in a minority of gastric carcinomas during carcinogenesis and progression, and suggest the proteasome-dependent pathway as a mechanism of PML protein loss.

Loss of caspase-1 gene expression in human gastric carcinomas and cell lines.

The caspases are a family of aspartic acid-specific proteases that fulfill varied and often critical roles in mammalian apoptosis or in the proteolytic activation of cytokines. Caspase-1 (interleukin-1beta-converting enzyme) is a member of the cysteine protease family, which cleaves target proteins following aspartic acid residues. We investigated caspase-1 expression in stomach cancer, tissues and cell lines. Of 301 consecutive gastric carcinomas, 58 cases (19.3%) showed the expressional loss of caspase-1. Loss of caspase-1 expression was significantly associated with pTNM stage (p=0.03), lymph node metastasis (p=0.01) and patient survival (p<0.01). Caspase-1 expression was also significantly correlated in an inverse manner with p53 expression (p<0.01). Among the 11 gastric cancer cell lines examined, three cell lines showed loss of expression at the protein and mRNA levels. On treatment with 5-aza-2'-deoxycytidine (5-aza-C), and/or trichostatin A (TSA), all three cell lines re-expressed caspase-1 mRNA. The above findings suggest that epigenetic events such as DNA methylation and histone deacetylation play important roles in the regulation of caspase-1, and that loss of caspase-1 expression is associated with poor survival in gastric carcinoma.

Mutation and altered expression of beta-catenin during gallbladder carcinogenesis.

Gallbladder carcinoma has two main morphologic developmental pathways: a dysplasia-carcinoma sequence and an adenoma-carcinoma sequence. beta-Catenin is a key regulator of the cadherin-mediated cell adhesion system, and altered expression and mutation of beta-catenin have been identified in many human malignancies. To clarify its role in gallbladder carcinogenesis, we investigated mutation and immunohistochemical expression of beta-catenin in adenomas, dysplasias, and carcinomas. We classified adenomas according to the expression of apomucins and cytokeratin and compared the mutational and expression pattern among each type. beta-Catenin mutations were identified in 58% (14 of 24) of the adenomas, and they were absent from all carcinomas (37 cases) and dysplasias (13 cases). Altered expression of beta-catenin, such as nuclear or cytoplasmic expression and loss of membranous expression, was also significantly higher in adenomas than in dysplasias or carcinomas (p <0.001). Of the adenomas, papillary adenomas and tubular adenomas of intestinal type showed infrequent beta-catenin abnormality, which was similar to the carcinomas. The cytoplasmic and nuclear expression of beta-catenin in carcinomas was correlated with less aggressive tumor behavior; in particular, cytoplasmic expression was associated with improved patient outcome (p = 0.028). Gallbladder adenoma may be a heterogeneous entity, and the majority of adenomas are not responsible for carcinoma progression.