Search

Oxidative stress is accepted as one of the main factors involved in the development and progression of cancer. Superoxide dismutases (SODs) are important in avoiding oxidative stress by eliminating reactive oxygen species (ROS). To determine whether single nucleotide polymorphisms at G7958A within SOD1 and at T5482C within SOD2 are associated with an increased susceptibility to gastric cancer, we investigated the genotype and allele frequencies of the genes from 294 gastric cancer patients and 300 healthy individuals. A polymerase chain reaction-single strand conformation polymorphism assay was used to identify the SOD1 G7958A and the SOD2 T5482C genotypes. Statistically significant differences in the genotype and allele frequencies of SOD2 T5482C were found between the healthy controls and gastric cancer patients (p = 0.0001 and p < 0.0001, respectively). When the data were stratified according to gastric cancer histological subtypes, the risk of both diffuse- and intestinal-type gastric cancer was statistically higher for carriers of the C allele compared with carriers of the T allele. However, there were no statistically significant differences in genotype distribution (p = 0.5069) and allele frequencies (p = 0.3714) of SOD1 G7958A between gastric cancer patients and controls. Our findings suggest that polymorphism of the SOD2 T5482C may be closely associated with an increased susceptibility to the development and differentiation of gastric cancer in the Korean population.

The aim of this study was to determine whether a single nucleotide polymorphism at G473A (rs1800449) within the LOX-propeptide is associated with susceptibility to gastric cancer. We investigated the genotype and allele frequencies of this gene in tissue specimens from 458 gastric cancer patients and 282 healthy individuals. Polymorphism analysis was performed by amplifying the propeptide region of LOX and digestion with NotI followed by sequencing of the products. The frequencies of the LOX G473A G/G, G/A, and A/A genotypes were 54.4% (249/458), 34.3% (157/458), and 11.3% (52/458), respectively, in gastric cancer patients and 58.9% (166/300), 35.5% (100/282), and 5.7% (16/282), respectively, in the healthy controls. Statistically significant differences in the genotype and allele frequency of LOX rs1800449 were observed between the healthy controls and gastric cancer patients (p = 0.0294 and p = 0.0339). When the data were stratified according to gastric cancer histologic subtype, the risk of diffuse-type gastric cancer in carriers with an A allele (G/A or A/A genotypes) was statistically higher compared to that of carriers with the G/G genotype (p = 0.0001). Our findings suggest that G473A polymorphism of the LOX gene may be closely associated with susceptibility to the development and differentiation of gastric cancer in South Korean patients., (© 2011 The Authors. APMIS © 2011 APMIS.)

Gastric atrophy and intestinal metaplasia are generally considered precancerous lesions of the stomach; Cdx2 plays an important role in intestinal metaplasia and gastric carcinogenesis. To elucidate the potential etiological role of the Cdx2 gene in gastric carcinogenesis, we analyzed genetic mutations and allelic loss in the Cdx2 gene of 95 sporadic gastric cancers. We found two somatic missense mutations in the Cdx2 gene, P63L in exon 1 and E204K in exon 2, encoding the caudal-like protein activation region (codon 13-180) and the homeobox domain (codon 188-243) of the gene, in the gastric cancers. In addition, 9 (25.0%) of 36 informative cases showed allelic loss at D13S220 and/or D13S260. In 11 cases with a genetic alteration, Cdx2 nuclear staining was observed only in 8 cases of gastric mucosa with intestinal metaplasia. Loss or reduced expression of the Cdx2 gene in cancer cells was found in two cases with a somatic mutation and in three cases with LOH. Interestingly, all of the cases were intestinal-type gastric cancers. Thus, these results suggest that genetic alterations of the Cdx2 gene may contribute to the loss of Cdx2 expression and to the development of gastric cancer, especially in the intestinal-type.

KLF4, which is also known as the gut-enriched Kruppel-like factor, plays important roles during the proliferation and differentiation of gastrointestinal epithelial cells. A loss of KLF4 expression has been observed in human tumors, particularly in the gastrointestinal tract. In this study, the molecular basis of the KLF4 inactivation in gastric cancer was investigated by analyzing the somatic mutation, the allelic loss with two microsatellite markers, D9S53 and D9S105, and hypermethylation of the KLF4 gene in 47 gastric adenomas and 81 gastric adenocarcinomas. Mutational analysis revealed one mutation of the KLF4 gene in a diffuse-type advanced gastric adenocarcinoma, but not in the gastric adenoma. This mutation was a somatic missense mutation, GGG-->AGG (Gly-->Arg) at codon 107 in exon 3, which encodes a transcriptional activation domain of the protein. An allelic loss was found in 7 (22.6%) of the 31 informative gastric adenoma cases and 15 (31.3%) of the 48 informative cancer cases at one or both markers. In addition, promoter hypermethylation of the KLF4 gene was observed in only two gastric cancers. These results suggest that genetic and epigenetic alterations of the KLF4 gene might play a minor role in gastric carcinogenesis.

EPHB2 is a member of the Eph receptor tyrosine kinase family and a direct transcriptional target of beta-catenin/TCF. EPHB2 plays an important role in maintaining the correct positioning of the proliferative compartment in the crypt-villous axis. A loss of EPHB2 expression has been observed in human tumors, particularly in colonic adenomas and carcinomas. A search was made for mutations at the A9 tract in exon 17, an allelic loss at the EPHB2 gene locus, and promoter hypermethylation of the EPHB2 gene in 81 sporadic gastric cancers in order to determine if genetic or epigenetic alterations of the EPHB2 gene are involved in the development and/or progression of gastric cancer. Unexpectedly, no frameshift mutation was found and there was a low frequency (20.8%) of allelic loss. In addition, promoter hypermethylation was detected in only one gastric cancer tissue sample. Therefore, genetic or epigenetic alterations of the EPHB2 gene might be an uncommon event in the development or progression of gastric cancers.

Beta-TrCP is a component of the ubiquitin ligase complex targeting beta-catenin for proteasomal degradation, and is a negative regulator of Wnt/beta-catenin signaling. To determine whether genetic alterations of the beta-TrCP gene are involved in the development or progression of gastric cancer, we analyzed its somatic mutations in 95 gastric cancers by single-strand conformational polymorphism and sequencing. We found five missense mutations (5.3%): A99V, H342Y, H425Y, C206Y, and G260E. Tissue carrying mutations showed moderate to strong cytoplasmic and/or nuclear staining of beta-catenin by immunohistochemistry. Thus, somatic mutations of the beta-TrCP gene may contribute to the development of gastric cancer through beta-catenin stabilization.

A recent report revealed that the protein-tyrosine phosphatase, receptor-type, T (PTPRT) gene is somatically mutated in several types of human cancer, suggesting that the mutated PTPRT gene is a tumor suppressor gene in human cancers. However, because previously the mutational search has focused primarily on colon cancers, data on PTPRT mutations in other types of human cancer have largely been lacking. Here, we performed a mutational analysis of the PTPRT phosphatase domain by polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) assay in 345 cases of common human cancers, including colon carcinomas, hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas and non-small cell lung cancers. We detected PTPRT phosphatase domain mutations in 1 of 105 colon carcinomas (1%) and 1 of 48 gastric carcinomas (2%), but none in acute leukemias, hepatocellular carcinomas, breast carcinomas and non-small cell lung cancers. The PTPRT mutation detected in the colon carcinoma was a missense mutation and the mutation in the gastric carcinomas was a splice-site mutation. Contrary to the previous report on the frequent PTPTR phosphatase domain mutations in colon cancers, this study demonstrated that the somatic mutation of the PTPRT phosphatase domain rarely occurred in common human cancers. The data suggested that alterations of the PTPRT-mediated signaling pathway by PTPRT phosphatase domain mutation may not play a critical role in the development of common human cancers.

Apoptosis-inducing factor (AIF) has a principal role in caspase-independent apoptosis. In addition, AIF has a vital oxidoreductase activity that is required for cell survival. It may be important to identify the alterations of the AIF gene and AIF protein expression to see the function of AIF in human cancers. In this study we analyzed the expression of AIF protein in 103 colorectal carcinomas by immunohistochemistry. We also analyzed the mutation in exons 10-15 of AIF encoding the region that possesses the cell death function of AIF by single-strand conformation polymorphism (SSCP) in 48 colorectal, 48 gastric, 48 breast and 48 hepatocellular carcinomas, and 48 leukemias. By immunohistochemistry, AIF protein expression was detected in both cancer cells and normal mucosal epithelial cells in all of the 103 colorectal carcinoma tissues. However, the cancer cells showed higher intensities of AIF immunostaining than the normal cells in 83 cases (80.5%). AIF immunoreactivities were observed in the cancers irrespective of their location or the depth of invasion. Mutational analysis detected one AIF mutation in the colorectal carcinomas, but none in the other cancers. The AIF mutation detected was a two-base deletion mutation in intron 15. The increased expression of AIF in the malignant colorectal epithelial cells compared to the normal mucosal epithelial cells suggests that AIF expression may play a role in colorectal tumorigenesis. The data also suggest that somatic mutation of AIF is a rare event in common human cancers.

Failure of apoptosis is one of the hallmarks of cancer. As an execution-phase caspase, caspase-6 plays a crucial role during apoptosis. To explore the possibility that the genetic alterations of CASP, which encodes caspase-6, might be involved in the development of human cancers, we analyzed the entire coding region and all splice sites of the human CASP6 gene for the detection of somatic mutations in 100 colorectal carcinomas and 50 gastric carcinomas. Overall, we detected three somatic mutations of the CASP6 gene, including two missense mutations and one splice-site mutation. The mutations were observed in two of the 100 colorectal carcinomas (2.0%) and one of the 50 gastric carcinomas (2.0%). Of note, one colorectal carcinoma with the CASP6 mutation harbored CASP3 and CASP8 gene mutations as well. We also analyzed caspase-6 expression by immunohistochemistry, and found that caspase-6 was expressed in 60% of the gastric cancers and 90% of the colorectal cancers. This is the first report on CASP6 gene mutations in human cancers, and these data indicate that the CASP6 gene is occasionally mutated in gastric and colorectal carcinomas. Also, the data suggest the possibility that deficiency of caspase-6 expression might contribute to the pathogenesis of gastric cancers.

The peptidyl-prolyl isomerase Pin1 is strikingly overexpressed in human cancers and is a novel regulator of beta-catenin. To determine whether somatic mutation of the Pin1 gene is involved in the development and/or progression of gastric cancer, we searched for mutations of the Pin1 gene in 95 gastric cancer specimens. The effect of Pin1 on beta-catenin expression was further examined in wild- and mutant-type Pin1-transfected HEK 293T cells. We found only one missense mutation that led to the substitution of alanine by aspartic acid at codon 118 of the Pin1 gene. On transfection study, the mutant Pin1 showed an increased expression of beta-catenin. However, the mutation had no effect on expression of the Pin1 protein in the case with Pin1 mutation. These results suggest that Pin1 may not play a role in the development or progression of gastric cancer.

In addition to chromosomal translocation, the MYC gene in the N-terminal coding region showed clustered point mutations in Burkitt's lymphomas. A recent study showed that these point mutations inactivated the apoptosis activity of MYC and contributed to the tumor transformation. To see whether the point mutations of MYC are involved in tumors besides Burkitt's lymphomas, we analyzed the N-terminal cluster region of the mutations in 507 cancers from gastric, colorectal, breast and lung carcinomas, and acute leukemias, by polymerase chain reaction-based single-strand conformation polymorphism assay. However, there was no somatic mutation of the MYC gene in the cancers. The data suggest that the point mutation of the MYC gene in the N-terminal domain may be very rare and may not contribute to the development of common human cancers besides Burkitt's lymphomas.

To investigate whether the KLF6 gene plays an important role in the development and/or progression of colorectal cancers, we searched for mutations and allelic loss of the KLF6 gene in 123 colorectal adenocarcinomas by performing PCR-SSCP sequencing. We found five somatic missense mutations: S155N, G163S, G163D, P183L and G195S. Three of them affected the activation domain of KLF6 and four mutations were predicted to disrupt the putative phosphorylation sites. On LOH analysis, 63 cases were heterozygous for at least one marker and 27 cases (42.9%) showed allelic loss at these markers. These data further support that the KLF6 gene may be one of the candidate tumor suppressor genes in colorectal cancers and that genetic alteration of the KLF6 gene might play a role in the development of colorectal carcinomas.

Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Caspase-9 plays a crucial role in the initiation phase of the intrinsic apoptosis pathway. To explore the possibility that the genetic alterations of caspase-9 might be involved in the development of human cancers, we analyzed the entire coding region and all splice sites of the human caspase-9 gene for the detection of somatic mutations in a series of 353 cancers, including 180 gastric, 104 colorectal and 69 lung adenocarcinomas. Overall, we detected three somatic mutations of caspase-9, but all of the mutations were silent mutations. The mutations were observed in 2 of 104 colorectal carcinomas and 1 of 180 gastric carcinomas. These data indicate that the caspase-9 gene is rarely mutated in gastric, colorectal and lung adenocarcinomas, and suggest that caspase-9 gene mutation may not contribute to the pathogenesis of these cancers.

CDX2 is a caudal-related homeobox transcription factor whose expression in the adult is normally restricted to the intestinal epithelium; it is implicated in the development and maintenance of the intestinal mucosa. The specific aim of this study was to elucidate the potential etiological role of CDX2 protein in colorectal carcinogenesis. We have analyzed the expression pattern of CDX2 protein in relation to the phenotype of 123 sporadic colorectal cancers by immunohistochemistry using tissue microarray. Strong CDX2 immunostaining was seen in the nuclei of corresponding normal intestinal epithelium. Interestingly, loss of CDX2 immunostaining was observed in 29 (23.6%) of 123 colorectal adenocarcinomas and its expression was correlated with the differentiation grade of the carcinoma (Chi-Square test, p<0.01). Clinically, CDX2 protein was immunopositive in 11 (91.7%) of 12 cases corresponding to stage A, 40 (85.1%) of 47 corresponding to stage B, 39 (69.6%) of 56 corresponding to stage C, and 4 (50.0%) of 8 corresponding to stage D. Statistically, CDX2 protein expression was related to tumor stage (Bartholomew test, p<0.05) and lymph node metastasis (Chi-Square test, p<0.05). These results indicate that loss of expression of CDX2 protein may play an important role in the tumorigenesis of colorectal cancers and that CDX2 expression represents a highly significant marker, which is able to identify a subset of patients at high risk.

ERBB2 is a member of the epidermal growth factor receptor (EGFR) family. Recent studies revealed that the kinase domain of the ERBB2 gene was mutated in human cancers, including gastric cancer. Despite the importance of cancer metastasis in the pathogenesis of cancers, data on the ERBB2 kinase domain mutation in cancer metastasis are lacking. In this study, to explore the possibility that ERBB2 mutation is involved in the metastasis mechanism, we analyzed the kinase domain of ERBB2 for the detection of somatic mutations in 58 gastric adenocarcinomas with lymph node metastasis. We found one ERBB2 mutation, which was detected in the lymph node metastasis, but not in the primary tumor of the same patient. The ERBB2 mutation was a missense mutation which substituted an amino acid in exon 21 (V832I). We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA and BRAF genes in the sample with the ERBB2 mutation, and found that this metastatic carcinoma did not harbor any of the mutations. Our data suggest that ERBB2 kinase domain mutation occasionally occurs in metastatic gastric carcinoma and might play a role in the metastatic process of some gastric carcinomas.

Accumulated evidence has established that aberrant regulation of histone deacetylases (HDACs) is one of the major causes of the development of human malignancies. Among different iso-enzymes of HDAC and sirtuins grouped as the HDAC super family, little is known as to how histone deacetylase 2 (HDAC2) causes carcinogenesis in solid tumors. Here, in order to investigate the possible role of HDAC2 in gastric carcinogenesis, we analyzed the expression of HDAC2 in 71 gastric adenocarcinomas by immunohistochemistry. Moderate to strong expression of HDAC2 was found in 44 (62%) out of a total of 71 tumors. The majority of positive tumors, which were detected in the nucleus but not in normal gastric epithelium, did not express HDAC2 or showed only weak positive staining. Interestingly, we also noted that HDAC2 expression appeared to be associated with tumor aggressiveness as HDAC2 expression was observed to be statistically significant in advanced gastric cancer (P=0.0023, Chi-square test) and in positive lymph node metastasis (P=0.0713, Chi-square test). Taken together, these results suggest that HDAC2 may play an important role in the aggressiveness of gastric cancer.

Deregulation of RAS signal transduction has been implicated in the malignant growth of human cancer cells. The BRAF gene, encoding a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers, raising the possibility that other RAF family members might be mutated in human cancers. In this study we analyzed the genomic DNAs for the detection of somatic mutations of the ARAF gene in 60 human cancer cell lines and 323 primary human cancer tissues, including colorectal carcinomas, gastric carcinomas, ovarian tumors and acute leukemias. The MOLT-4 leukemia cell line was found to harbor an ARAF gene mutation resulting in an amino acid substitution (A451T) at the activation segment in the kinase domain of ARAF. In the cancer tissues we could not detect any ARAF gene mutation. Our data indicate that, in contrast to the BRAF gene, the ARAF gene is rarely mutated in human cancers, and suggest that alterations of the RAS pathway by ARAF gene mutation may not play an important role in the pathogenesis of human cancers.

Failure of apoptosis is one of the hallmarks of cancer. As an adaptor, FADD plays a crucial role during death receptor-mediated apoptosis. We previously reported that the FADD gene is somatically mutated in non-small cell lung cancers. To explore the possibility that the genetic alterations of the FADD gene might be involved in the development of other human cancers, we analyzed the entire coding region and all splice sites of the human FADD gene to detect somatic mutations in 116 stomach cancers and 98 colon cancers. Overall, we detected a somatic missense mutation of the FADD gene in a colon carcinoma, which was predicted to change an amino acid (R140H) in the death domain (DD) of the FADD protein. This is the first report of FADD gene mutation in gastrointestinal cancers, and our data suggest that the FADD gene is rarely mutated in human colon and stomach cancers.

K(+) channels have been reported to be involved in the proliferation of many types of cells, including some human carcinoma and tumor cell lines. KCNK9, a TASK channel, is amplified and overexpressed in several types of human cancer. In the present study, we examined the expression and somatic mutations of KCNK9 in 124 colorectal cancers by immunohistochemistry using tissue microarray and PCR-SSCP. Immunopositivity was observed in 57 (46.0%) of 124 colorectal cancers. Clinically, KCNK9 was immunopositive in 4 (30.7%) of 13 cases which were stage A, 26 (55.3%) of 47 which were stage B, 23 (41.1%) of 56 which were stage C, and 4 (50%) of 8 which were stage D. Statistically, KCNK9 protein expression was not related to tumor stage (Bartholomew test, p>0.05) and lymph node metastasis (Chi-Square test, p=0.8338). In the mutation study of the KCNK9 gene, we found only one sequence variation (ACG-->ACC, Thr-->Thr) at codon 170 both in corresponding normal and tumor DNAs. These results indicate that overexpression rather than mutation of the KCNK9 gene may contribute to the development of colorectal cancers and suggest that the development of KCNK9-targeted agents may provide new possibilities in the treatment of colorectal cancer.

Caspases play an essential role during apoptotic cell death, and alterations of caspases are known to contribute to human cancer development. In the current study, we analyzed the expression of caspase-2, -6 and -7 in 120 gastric carcinomas by immunohistochemistry using a tissue microarray approach. Caspase-2, -6 and -7 were expressed in 42 (35%), 63 (53%) and 39 (33%) of the gastric cancers, respectively. By contrast, the surface mucous cells and mucosal glandular cells in the normal gastric mucosa showed strong immunoreactivity for caspase-2, -6 and -7. Taken together, these results indicate that caspase-2, -6 and -7 expression in gastric cancer cells is decreased compared to in normal gastric mucosal cells, and suggest that loss of caspase-2, -6 and -7 expression might be involved in the mechanisms of gastric cancer development.

Mounting evidence suggests that activation of the mitogen-activated protein (MAP) kinase pathway plays an important role in tumorigenesis. MAP kinase/ERK kinase (MEK), a crucial constituent of this pathway, is activated by phosphorylation, and the phosphorylated MEK (pMEK) in turn activates ERK kinase. The expression of pMEK has been described in some human malignancies, but not in primary human colon tumors. In this study, we analyzed the expression of pMEK in 123 colorectal tumors by immunohistochemistry. pMEK was detected either in the cytoplasm (63 cases) or nucleus (40 cases) in 93 of the 123 tumors (76%). Tubular adenomas and villous adenomas also expressed pMEK in 30% and 40% of the tumors, respectively. By contrast, the epithelial cells in the normal colonic mucosa showed no or only weak expression of pMEK in the cytoplasm. Taken together, these results indicate that MEK is frequently phosphorylated in colorectal tumors, and suggest that phosphorylation of MEK may play a role in the development of colorectal tumors.

It is believed that clonal expansion and cancer growth is the result of the deregulation of proliferation and cell death. Recently, salvador, a molecule that can regulate both cell proliferation and cell death, was identified. It was also reported that human salvador (hWW45) is mutated in some cancer cell lines. However, there have been no data regarding salvador gene mutations in human cancer tissues. To explore the hypothesis that the salvador gene might be similarly mutated in human cancer tissues, we analyzed the entire coding region of the salvador gene for the detection of somatic mutations in a series of human cancer tissues, including carcinomas from stomach, colon, liver and lung. However, using SSCP analysis, no mutation in the coding and splicing regions could be detected in the cancers. The data presented here suggest that salvador is not frequently mutated in human carcinoma tissues and that the mutation might be tumor-type specific.

Elevated levels of the calcium-binding protein S100A4 cause metastasis of benign rat mammary tumor cells. To investigate whether S100A4 plays an important role in the invasion and metastasis of gastric cancers, we examined the gene mutations in the coding regions and expression patterns of the S100A4 in gastric adenocarcinoma in Korea. Moderate to strong expression of S100A4 was found in 53 (68.8%) of the 77 gastric adenocarcinomas, whilst normal gastric epithelium either failed to stain or showed weak staining. Interestingly, S100A4 expression was more frequently observed in gastric cancer patients with advanced gastric cancer (p=0.039), positive lymph node metastasis (p=0.001), and peritoneal dissemination (p=0.022). No gene mutations were found in the analyzed genomic area in 77 gastric adenocarcinomas and 15 gastric cancer cell lines. We found one single nucleotide polymorphism without an amino acid change, A99G, in two cases. These data suggest that the overexpression of S100A4 may be closely related to the aggressiveness of gastric cancer in Korea.