Jae Hwi Song

Inactivation of the Gastrokine 1 gene in gastric adenomas and carcinomas

Gastrokine 1 (GKN1) plays a role in the gastric mucosal defence mechanism and may be a gastric tumour suppressor. We have investigated whether inactivation of the GKN1 gene is involved in the development and/or progression of gastric cancers. GKN1 protein expression was examined in gastric adenomas and cancer and we also analysed GKN1 mutation and epigenetic alteration, DNA copy number change and mRNA transcript expression. The effect of GKN1 on cell proliferation and death was examined in wild‐type GKN1‐transfected AGS gastric cancer cells. Reduced or loss of GKN1 expression was detected in 36 (90%) and 170 (89.5%) of 40 adenomas and 190 gastric cancers, respectively. Statistically, there was no significant relationship between altered expression of GKN1 protein and clinicopathological parameters, including depth of invasion, location and lymph node metastasis (χ2 test, p > 0.05). In western blot analysis, absence or reduced expression was found in 21 (84.0%) of 25 gastric carcinomas. No mutation was detected in gastric tumours, and hypermethylation of GKN1 gene was found in two tumours. DNA copy number and mRNA transcript of GKN1 were significantly decreased in gastric cancers. In functional analysis, AGS gastric cancer cells transfected with GKN1 wild‐type showed marked inhibition of cell proliferation and induction of cell death. These data suggest that inactivation of the GKN1 gene may play an important role in the development of sporadic gastric cancers, as an early event. Copyright

Genetic Alterations of the ATBF1 Gene in Gastric Cancer

Purpose: α-Fetoprotein (AFP)–producing gastric cancers are aggressive tumors with venous and lymphatic invasion and hepatic metastasis. The goal of the present study was to investigate whether somatic changes of the AFP-negative regulator AT motif binding factor-1 (ATBF1) gene are involved in the development or progression of gastric cancers and the production of AFP in gastric cancer cells. Experimental Design: We searched for genetic alterations of the ATBF1 gene by single-strand conformational polymorphism and sequencing methods as well as allelic loss analysis with the microsatellite markers D16S3066 and D16S3139. Immunochemistry for AFP expression in gastric cancer cells was also done. Results: In 81 sporadic gastric cancers, four mutations were detected in seven cases: one was a missense mutation and three were deletions; loss of heterozygosity at the ATBF1 locus was detected in 52.9% of informative samples. Five of the eight cancers with AFP expression showed ATBF1 genetic alterations. Conclusions: These results suggest that genetic alteration of the ATBF1 gene may contribute to the aggressive nature of gastric cancers and the production of AFP in gastric cancer cells.

Somatic mutations of the β-TrCP gene in gastric cancer†

Beta‐TrCP is a component of the ubiquitin ligase complex targeting β‐catenin for proteasomal degradation, and is a negative regulator of Wnt/β‐catenin signaling. To determine whether genetic alterations of the β‐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 β‐catenin by immunohistochemistry. Thus, somatic mutations of the β‐TrCP gene may contribute to the development of gastric cancer through β‐catenin stabilization.

Genetic and epigenetic analysis of the EPHB2 gene in gastric cancers

EPHB2 is a member of the Eph receptor tyrosine kinase family and a direct transcriptional target of β‐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.

Altered expression of CDX2 in colorectal 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.

Aspartic acid repeat polymorphism of the Asporin gene with susceptibility to osteoarthritis of the knee in a Korean population

Osteoarthritis (OA) is a common disease that is characterized by the degeneration of joint cartilage in the knee, hip and hand. Recently, it has been reported that susceptibility to OA is affected by the number of aspartic acid (D) residues in the amino-terminal of the asporin protein. In order to investigate whether the association found in Japanese OA is also present in the Korean population, the frequency of the polymorphism was examined in 190 OA patients and 376 healthy controls. The D-repeat microsatellite polymorphism was examined by amplifying the asporin gene and sequencing its products. The frequencies of the D13 and D14 alleles were 69.7% (265/380) and 5.8% (22/380), respectively, in OA patients and 64.2% (483/752) and 8.7% (65/752), respectively, in healthy controls. When we adjusted for gender and age, there was no statistically significant difference between Korean OA patients and healthy controls (P=0.1082) in the allele frequency of D13 compared to the other alleles. However, a significant difference between female OA patients and their controls (P=0.0245) in the allele frequency of the D13 allele was found compared to the other alleles. There was no significant difference between Korean OA patients and healthy controls in the frequency of the D14 and other alleles (P=0.2339). These results suggest that asporin may play a role in OA susceptibility of the knee in the Korean female population.

Activation-Induced Cytidine Deaminase Expression in Gastric Cancer

Helicobacter pylori increases the risk of gastric cancer development and triggers aberrant expression of activation-induced cytidine deaminase (AID). The goal of the present study was to investigate whether AID expression is involved in the development or progression of gastric cancer and the nuclear expression of p53 protein in cancer cells. We examined the expression pattern of the AID and p53 proteins in 186 gastric adenocarcinomas by immunohistochemistry. In order to investigate the molecular mechanism of AID expression, we also searched for mutations in the AID gene by single-strand conformational polymorphism and sequencing methods. In 186 sporadic gastric cancers, AID expression was detected in the 73 corresponding normal gastric mucosa and in 50 gastric cancers. Statistically, the expression of AID protein was not associated with clinicopathological parameters, including tumor size, location, differentiation and lymph node metastasis (p > 0.05). Interestingly, a significant association was observed between AID and the nuclear expression of p53 (p = 0.0094). Mutational analysis revealed no mutation in the AID gene in the gastric cancers. These results suggest that aberrant expression of the AID protein may contribute to the development of gastric cancers and induce p53 nuclear expression.

Genetic analysis of the DBC2 gene in gastric cancer

The DBC2 (Deleted in breast cancer, RhoBTB2) has been identified as a tumor suppressor gene that has growth inhibitory function. To investigate whether genetic alterations of the DBC2 gene are involved in the development of gastric cancer, we analyzed mutations and allelic loss in the DBC2 gene in 95 primary gastric cancers by PCR-SSCP, sequencing and LOH analysis. In the mutational analysis, we found one missense somatic mutation (CGG→TGG, R275W) in the BTB/POZ domain of the gene in a patient with advanced gastric cancer and lymph node metastasis. In addition, we found one known polymorphism and three novel polymorphisms in the coding region of DBC2, which showed an amino acid change, and was detected in both the cancer cells and corresponding normal cells. On LOH analysis, 62 cases were heterozygous for at least one marker and 18 cases (29.0%) showed allelic loss at these markers. In conclusion, the mutations and allelic loss in the DBC2 gene are uncommon in gastric cancers in Korean patients. Further studies to identify the target gene at 8q21 responsible for the development of gastric cancer should be explored.

Overexpression of serine–threonine receptor kinase-associated protein in colorectal cancers

Transforming growth factor‐β (TGF‐β) regulates many cellular processes, including cellular proliferation and differentiation. Disruption of the TGF‐β signaling pathway can lead to cancer. Serine–threonine receptor kinase‐associated protein (STRAP), an inhibitor of TGF‐β signaling, is an important regulator of cell proliferation. Here, in order to investigate the roles of STRAP in colorectal carcinogenesis, the expression of the STRAP protein was investigated in 59 colonic adenomas and 123 colorectal cancers by immunohistochemistry. Upregulation of STRAP protein was observed in 30 (50.8%) of 59 adenomas and 87 (70.7%) of 123 cancers, respectively. Statistically, overexpression of STRAP protein was not associated with clinicopathological parameters and 5 year survival (P > 0.05). Interestingly, significant association was observed between STRAP and Ki‐67 positivity (P < 0.05), suggesting that STRAP contributes to an increased proliferate potential of tumor cells. These results indicate that upregulation of STRAP might play a role in tumor development as an early event for colorectal cancers.

Genetic alterations and expression pattern of CEACAM1 in colorectal adenomas and cancers.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed on epithelial cells throughout the intestinal tract and is a negative regulator of tumor cell growth, suggesting that it may function as a tumor suppressor. In this study, to determine whether the CEACAM1 is involved in colorectal tumorigenesis, we have investigated the genetic alterations, including mutations and allelic loss, of the CEACAM1 gene in 17 colonic adenomas and 123 sporadic colorectal cancers. In addition, the expression pattern of the CEACAM1 protein was examined in 60 colonic adenomas and 123 sporadic colorectal adenocarcinomas. No mutation was found in colonic adenomas, but four somatic missense mutations, L36F, T312I, V398I and A445V, were detected in colorectal cancers. Interestingly, all of the mutations were found in left-side colon cancers of the patients with clinical stage III. In LOH analysis, nine adenomas were informative for at least one of the markers and five (55.6%) showed allelic loss. Thirty-eight cancers were informative at D19S211 and D19S872 markers and 21 (56.3%) showed LOH at these markers. Statistically, the frequency of allelic loss at the CEACAM1 locus was not associated with clinicopathologic parameters (P > 0.05). In immunohistochemical analysis, loss of expression of CEACAM1 protein was detected in nine (15.0%) and 30 (24.4%) of 60 colorectal adenomas and 123 colorectal cancers. Statistically, there was no significant relationship between loss of CEACAM1 expression and clinicopathologic parameters, including clinical stage, tumor location, tumor size, lymph node metastasis and 5-year survival (P > 0.05). These data suggest that genetic alteration and loss of expression of the CEACAM1 may contribute to the development of colorectal cancers, as an early event.