Keisuke Matsusaki

DNA methylation of multiple genes in gastric carcinoma: Association with histological type and CpG island methylator phenotype

Hypermethylation of CpG islands is associated with silencing of various tumor suppressor genes. Recent studies on colorectal and gastric cancer have identified a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. For determination of association between DNA methylation pattern or histological type and CIMP status in gastric carcinoma, CpG islands in the promoters of hMLH1 and CDH1 genes, CpG islands overlapping exon 1 of MGMT and p16INK4a genes, and a non‐CpG island in exon 1 of the RAR‐β gene were studied. The presence of the CIMP was determined by monitoring five methylated in tumor (MINT) loci in 103 gastric carcinomas. Among the 103 gastric carcinomas, DNA hypermethylation was detected in the following frequencies: 14 (14%) for hMLH1, 26 (25%) for MGMT, 26 (25%) for p16INK4a, 54 (52%) for CDH1, and 53 (52%) for RAR‐β. Forty‐two (41%) of 103 gastric carcinomas were positive for the CIMP. CIMP and hypermethylation of p16INK4a gene were found more frequently in intestinal and diffuse‐adherent types than in diffuse‐scattered type (P=0.013 and 0.017, respectively). In contrast, hypermethylation of the CDH1 and RAR‐β genes was more common in the diffuse‐scattered type than in the other types (P=0.008 and 0.007, respectively). In intestinal‐ and diffuse‐adherent‐type gastric carcinomas, we found significant associations between the presence of the CIMP and hypermethylation of several genes: hMLH1 (P=0.006), p16INK4a (P=0.018), CDH1 (P=0.024), and RAR‐β (P=0.044). Our overall results suggest that in some intestinal‐ and diffuse‐adherent‐type gastric carcinomas, DNA hypermethylation affects non‐specific gene promoters concordantly, at least in part, whereas in diffuse‐scattered‐type gastric carcinoma, DNA hyper‐methylation affects specific genes such as CDH1 and RAR‐β.

A single nucleotide polymorphism in the MMP-9 promoter affects tumor progression and invasive phenotype of gastric cancer

Purpose Matrix metalloproteinase-9 (MMP-9, gelatinase B) plays a key role in cancer invasion and metastasis by degradating the extracellular matrix (ECM) and basement membrane barriers. A cytosine (C)-thymidine (T) single nucleotide polymorphism (SNP) at position –1562 in the MMP-9 promoter is reported to affect expression of this gene. The purpose of this study was to investigate the relation between the –1562 C/T polymorphism and the development and progression of gastric cancer.Methods The study population included 177 gastric cancer patients and 224 healthy control subjects. The SNP in the MMP-9 promoter was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing. Genotype frequencies were compared between patients and controls, and the association of genotypes with clinicopathological features was studied.Results Genotype frequencies in gastric cancer patients were similar to those in control subjects (P = 0.223). However, significant association was found between degree of tumor invasion, clinical stage, and lymphatic invasion and the MMP-9 polymorphism in gastric cancer patients (P<0.05, for each).Conclusions Our results indicate that the T allele in the MMP-9 promoter is associated with the invasive phenotype of gastric cancer.

Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas.

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation‐specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16INK4a, cadherin 1 (CDH1), and retinoic acid receptor‐beta (RAR‐beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR‐beta promoter hypermethylation is associated with CIMP‐positive gastric carcinoma. CpG island hypermethylation of the p16INK4a, CDH1, and RAR‐beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16INK4a promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fishers exact test) and was inversely associated with p53 mutations (p = 0.0225; Fishers exact test). However, CDH1 and RAR‐beta promoter hypermethylation was observed more frequently in diffuse–scattered type gastric carcinoma than in other types (intestinal and diffuse–adherent types) (p = 0.0175 and p = 0.0335, respectively; Fishers exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR‐beta promoters occurred concordantly (p < 0.0001; Fishers exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16INK4a promoter hypermethylation) and diffuse–scattered types (CDH1 and RAR‐beta promoter hypermethylation) of gastric carcinoma. Copyright

A Single Nucleotide Polymorphism in the 5′ Untranslated Region of the EGF Gene Is Associated with Occurrence and Malignant Progression of Gastric Cancer

Objective: Epidermal growth factor (EGF) has many biological functions and plays an important role in the progression of various tumors including gastric cancer. An A-G single nucleotide polymorphism (SNP) at position 61 in the 5′-untranslated region (UTR) of the EGF gene has recently been reported to be associated with different levels of EGF production. We examined whether this polymorphism is correlated with the development and malignant phenotypes of gastric cancer. Methods: The study population included 200 gastric cancer patients and 230 healthy control subjects. The SNP in the 5′-UTR of the EGF gene was analyzed by polymerase chain reaction-restriction fragment length polymorphism. Results: The A allele was significantly less frequent in patients than in controls (p = 0.01). Individuals with the A/A or A/G genotype showed a significantly lower risk of gastric cancer than those with the G/G genotype [adjusted odds ratio (OR) = 0.56], whereas the same genotypes were associated with malignant progression of this cancer, e.g. deeper tumor invasion, increased lymph node metastasis and advanced clinical stage, and histological classification in gastric cancer patients (adjusted OR = 1.80, 1.98, 2.26 and 1.89, respectively). Conclusions: Our findings suggest that the A-G polymorphism of EGF is involved not only in the occurrence but also in the malignant progression of gastric cancer.

Differential expression of claudin-2 in normal human tissues and gastrointestinal carcinomas

Claudins are involved in the formation of tight junctions in epithelial and endothelial cells. Claudins form a family of 24 members displaying organ- and tissue-specific patterns of expression. In the present study, we evaluated the specificity of the claudin-2 expression in various normal human tissues and gastrointestinal cancers by quantitative reverse transcriptase–polymerase chain reaction and immunohistochemistry. In 14 various normal tissues, claudin-2 mRNA was expressed in the kidney, liver, pancreas, stomach, and small intestine; the highest level of which was detected in the kidney. Colorectal cancers (CRCs) expressed claudin-2 mRNA at high levels. Immunohistochemical analysis of claudin-2 in 146 gastric cancers (GCs) and 99 CRCs demonstrated claudin-2 expression in 2.1% of GCs and 25.3% of CRCs, respectively. There was no obvious correlation between claudin-2 expression and clinicopathological parameters of CRCs. These results suggest that the expression of claudin-2 may involve organ specificity, and increased expression of claudin-2 may participate in colorectal carcinogenesis.

Reduced Expression of the TSP1 Gene and Its Association with Promoter Hypermethylation in Gastric Carcinoma

Thrombospondin-1 (TSP1) is a potent peptide shown in some tumor systems to be linked with angiogenesis. Epigenetic alteration of TSP1 has been reported in various primary tumors. However, the expression pattern of TSP1 has not been characterized in gastric carcinoma. We measured levels of TSP1 mRNA expression using quantitative RT-PCR in 30 gastric carcinomas and 10 non-neoplastic mucosae. In addition, we examined the correlation of the levels of TSP1 mRNA expression levels with promoter methylation status of TSP1 monitored by methylation-specific PCR as well as p53 mutation status detected by PCR-single-strand conformation polymorphism. Promoter hypermethylation of the TSP1 gene was found in 10 (33%) of 30 gastric carcinomas, and TSP1 mRNA expression levels were associated with promoter hypermethylation of TSP1 (p = 0.017; Mann-Whitney U test). p53 mutation was found in 5 (17%) of 30 gastric carcinomas, however, TSP1 mRNA expression was not associated with p53 mutation status (p = 0.858; Mann-Whitney U test). There was no correlation between TSP1 mRNA expression levels and T grade, N grade, tumor stage, or histological type. Our results suggest that transcriptional inactivation of TSP1 by aberrant DNA methylation of the promoter region may participate partly in stomach carcinogenesis through TSP1 down-regulation.

DNA hypermethylation and histone hypoacetylation of the HLTF gene are associated with reduced expression in gastric carcinoma

The SWI/SNF proteins are ATP‐dependent chromatin remodeling enzymes that have been implicated in the regulation of gene expression. Recent studies have shown that members of the SWI/ SNF superfamily can function as tumor suppressor genes. DNA methylation and transcriptional inactivation of the HLTF gene, which is a homologue to the SWI/SNF genes, have been observed in colon cancer. In the present study, we studied the DNA methylation status of the HLTF gene by methylation‐specific PCR in 50 gastric carcinoma tissues, and seven gastric carcinoma cell lines and compared the methylation status with the levels of HLTF mRNA expression. DNA methylation of the HLTF gene was found in 25 (50%) of 50 gastric carcinomas, and levels of HLTF mRNA were associated with methylation status of HLTF (P=0.027; Mann‐Whitney U test). No correlations were found between HLTF mRNA levels and DNA methylation and T grade, N grade, tumor stage, or histological type. In corresponding non‐neoplastic mucosae, DNA methylation of the HLTF gene was found in 1 (7%) of 15 samples. The methylated allele was not detected in any of 10 normal gastric mucosae from 10 healthy volunteers. Among seven gastric carcinoma cell lines, the KATO‐III cell line showed loss of HLTF mRNA expression associated with DNA methylation. This loss was rectified by treatment with both Aza‐2′‐deoxycytidine, a demethylating agent, and trichostatin A, a histone deacetylase inhibitor. Chromatin immunoprecipitation assay revealed that the acetylation levels of histones H3 and H4 in the 5’CpG island of the HLTF gene were inversely associated with DNA methylation status. These results suggest that transcriptional inactivation of HLTF by aberrant DNA methylation and histone deacetylation may be involved in stomach carcinogenesis through down‐regulation of HLTF expression.

A single nucleotide polymorphism in the transmembrane domain coding region of HER-2 is associated with development and malignant phenotype of gastric cancer

Alterations of the HER‐2 (erbB‐2/neu) proto‐oncogene have been associated with carcinogenesis and poor prognosis of certain cancers. A single nucleotide polymorphism (Ile/Val, A/G) in the transmembrane domain was reported to be associated with a risk of breast cancer. In our study, we examined the association between the HER‐2 polymorphism and gastric carcinoma. The Ile/Ile, Ile/Val and Val/Val genotypes were found in 146 (68.9%), 56 (26.4%) and 10 (4.7%) of 212 gastric cancer patients and in 234 (81.5%), 48 (16.7%) and 5 (1.8%) of 287 control subjects, respectively. The Ile/Val or Val/Val genotype was significantly more frequent in patients than in controls (p = 0.005 and 0.033, respectively). The OR of Val/Val genotype then revealed a significantly enhanced risk of 3.25 (95% CI 1.09–9.70) compared to Ile/Ile genotype; heterozygous Ile/Val genotype showed an intermediate risk of 1.97 (1.27–3.06). In patients, carcinomas of advanced stage were significantly more frequent in patients with Ile/Val or Val/Val genotype than those with Ile/Ile genotype (p < 0.001). The logistic regression analysis for tumor invasion, lymph node metastasis and distant metastasis revealed that lymph node metastasis was most closely associated with the HER‐2 genotype. These results suggest that this nucleotide polymorphism in the transmembrane domain‐coding region of HER‐2 could be associated with development of gastric carcinoma and may serve as a predictor of risk for a malignant phenotype of gastric cancer. The association of HER‐2 genotype with clinicopathologic characteristics of gastric cancer was also suggested, which has to be confirmed with a larger sample size.

Immunohistochemical analysis of colorectal cancer with gastric phenotype: Claudin-18 is associated with poor prognosis

Claudin‐18 plays a key role in constructing tight junctions, and altered claudin‐18 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin‐18 in colorectal cancer (CRC). The aim of this study is to investigate the significance of claudin‐18 expression in CRC and its association with clinicopathological factors. We performed clinicopathological analysis of claudin‐18 expression in a total of 569 CRCs by immunohistochemistry. Moreover, we investigated the association between claudin‐18 and various markers including gastric/intestinal phenotype (MUC5AC, MUC6, MUC2 and CD10), CDX2, claudin‐3, claudin‐4, p53 and Ki‐67.

Molecular characteristics of differentiated-type gastric carcinoma with distinct mucin phenotype: LI-cadherin is associated with intestinal phenotype

Gastric carcinomas (GC) are classified into four phenotypes on the basis of the mucin expression profile: G type (gastric or foveolar phenotype), I type (intestinal phenotype), GI type (intestinal and gastric mixed phenotype) and N type (neither gastric nor intestinal phenotype). Immunohistochemistry was used to examine the expression of epidermal growth factor receptor (EGFR), E‐cadherin, liver–intestine (LI)‐cadherin, CD44v9 and p53 and correlation of these molecules with mucin phenotype and tumor stage was evaluated. Overexpression of EGFR and LI‐cadherin, reduced expression of E‐cadherin and abnormal expression of p53 were observed more frequently in advanced GC than in early GC. Among I‐type GC, overexpression of EGFR and reduced expression of E‐cadherin were observed more frequently in advanced tumors than in early tumors. Among G‐type GC, reduced expression of E‐cadherin was significantly associated with advanced tumors. With respect to the relationship between mucin phenotype and expression of cancer‐related molecules, overexpression of LI‐cadherin was observed more frequently in I‐type (12/25, 48.0%) than in G‐type (1/14, 7.1%) GC. I‐type GC tended to express LI‐cadherin more frequently than GI‐type GC. These results provide insights into the molecular characteristics of the distinct mucin phenotype of differentiated‐type GC and suggest that LI‐cadherin may contribute to the biological behavior of I‐type GC.