Kazuya Kuraoka

Expression of receptors for advanced glycation end‐products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer

The receptor for advanced glycation end‐products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S‐oligodeoxynucleotide was nine times less than that of sense S‐oligodeoxynucleotide‐treated cells. Growth of cells under RAGE antisense S‐oligodeoxynucleotide treatment was not different from that seen under sense S‐oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non‐RAGE‐expressing cell line). RAGE antisense S‐oligodeoxynucleotide treatment suppressed the invasive activity of RAGE‐positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen‐coated membrane under RAGE antisense S‐oligodeoxynucleotide treatment was significantly lower than under RAGE sense S‐oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S‐oligodeoxynucleotide‐treated RAGE‐negative MKN45 cells showed no difference. A wound‐healing assay showed that no RAGE antisense S‐oligodeoxynucleotide‐treated MKN28 cells migrated into the scraped area, whereas sense S‐oligodeoxynucleotide‐treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE‐positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE‐positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer. Copyright

Search for new biomarkers of gastric cancer through serial analysis of gene expression and its clinical implications

Gastric cancer is one of the most common human cancers and is the second most frequent cause of cancer‐related death in the world. Serial analysis of gene expression (SAGE) is a powerful technique to allow genome‐wide analysis of gene expression in a quantitative manner without prior knowledge of the gene sequences. SAGE on 5 samples of gastric cancer with different histology and clinical stages have created large SAGE libraries of gastric cancer that enable us to identify new cancer biomarkers. Commonly up‐regulated genes in gastric cancer in comparison with normal gastric epithelia included CEACAM6, APOC1 and YF13H12. By comparing gene expression profiles of gastric cancers at early and advanced stages, several genes differentially expressed by tumor stage were also identified, including FUS, CDH17, COL1A1 and COL1A2, which should be novel genetic markers for high‐grade malignancy. Regenerating gene type IV (REGIV) is one of the most up‐regulated genes in a SAGE library of a scirrhous‐type gastric cancer. In vitro studies using RegIV‐transfected cells revealed that RegIV is secreted by cancer cells and inhibits apoptosis, suggesting that RegIV may serve as a novel biomarker and therapeutic target for gastric cancer. Production of RNA aptamers could be a useful approach to establish a detection system in blood. A custom‐made array, named Ex‐STO‐MACHIP, consisting of 395 genes, including highly differentially expressed genes identified by our SAGE and other known genes related to carcinogenesis and chemosensitivity, is useful to study the molecular pathogenesis of gastric cancer and to obtain information about biological behavior and sensitivity to therapy in the clinical setting. Combined analyses of gene expression profile, genetic polymorphism and genetic instability will aid not only cancer detection, but also characterization of individual cancers and patients, leading to personalized medicine and cancer prevention.

Adenocarcinoma arising from a gastric duplication cyst with invasion to the stomach: a case report with literature review

This report describes a rare case of adenocarcinoma arising from a gastric duplication cyst, with invasion to the stomach wall, in a 40 year old Japanese man. A cystic lesion was found between the stomach and the spleen. The cyst had a well circumscribed smooth muscle layer, corresponding to the muscularis propria of the stomach and the mucosa of the alimentary tract. A well differentiated adenocarcinoma was found within the duplication cyst, invading its serosa. Well differentiated adenocarcinoma was independently found in the fundus of the stomach; the tumour of the cyst was connected by fibrous tissue. Microscopically, there was neither adenocarcinoma in situ nor precancerous lesions, such as epithelial dysplasia, suggesting that the carcinoma derived from a gastric duplication cyst that invaded the stomach. Duplication cysts should be included in the differential diagnosis of cystic masses of the gastrointestinal tract, and the possibility of malignancy within these cysts should be considered.

Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma

Suppressor of cytokine signaling (SOCS)‐1 inhibits signaling of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway by several cytokines and has tumor suppressor activity. Methylation of the SOCS‐1 CpG island has been shown to inactivate the SOCS‐1 gene in certain human cancers. In our study, we investigated methylation status of the SOCS‐1 gene by methylation‐specific PCR in 75 gastric carcinoma (GC) tissues, 25 corresponding nonneoplastic mucosae and 10 normal gastric mucosae from healthy young individuals. We also performed bisulfite sequencing of DNAs from 2 GC tissues. In addition, SOCS‐1 mRNA levels were examined in 50 GCs by quantitative RT‐PCR. Hypermethylation of the SOCS‐1 gene was detected in 33 (44%) of 75 GC tissues and in 3 (12%) of 25 corresponding nonneoplastic mucosae; the incidence was significantly different (p = 0.004). None of the 10 normal gastric tissues from healthy individuals showed hypermethylation. Methylation of the SOCS‐1 gene was associated with lymph node metastasis, advanced tumor stage and reduced expression of SOCS‐1 in GC tissues (p = 0.009, 0.034 and 0.002, respectively). Reduced expression of SOCS‐1 in GC tissues was associated with lymph node metastasis and advanced tumor stage (p = 0.013 and 0.002, respectively). Our results suggest that transcriptional inactivation of the SOCS‐1 gene by hypermethylation may be involved in development, progression and metastasis of GC.

Promoter hypermethylation of MGMT is associated with protein loss in gastric carcinoma

Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes in neoplasms. Recently, O6‐methylguanine‐DNA methyltransferase, MGMT, was shown to be hypermethylated in certain carcinomas, resulting in loss of MGMT protein. We studied DNA methylation of CpG islands of the MGMT gene by methylation specific PCR in 26 gastric carcinoma tissues and 8 gastric carcinoma cell lines for comparison with levels of MGMT protein expression. In addition, we examined p53 mutation status in the same tissues by PCR‐SSCP analysis for comparison with MGMT protein expression levels. In total, promoter hypermethylation of the MGMT gene was found in 8 (31%) of the 26 gastric carcinomas with reduced expression of MGMT protein, whereas the hypermethylation was not detected in the 18 carcinomas with non‐reduced MGMT expression. MGMT protein expression levels were associated with promoter hypermethylation of MGMT (p = 0.0001; Mann‐Whitney test); however, MGMT expression was not associated with p53 mutation status (p = 0.461; Mann‐Whitney test). Among in gastric carcinoma cell lines, the TMK‐1 cell line showed loss of the MGMT protein association with promoter hypermethylation and this loss was rectified by treatment with a demethylating agent, 5‐Aza‐2′‐deoxycytidine. Our results suggest that transcriptional inactivation of MGMT by aberrant methylation of the promoter region may participate in carcinogenesis in the stomach.

Increased expression but not genetic alteration of BRG1, a component of the SWI/SNF complex, is associated with the advanced stage of human gastric carcinomas.

BRG1, a component of the SWI/SNF complex, regulates gene transcription through chromatin remodeling. Certain human cancer cell lines have been shown to contain homozygous deletions or mutations, half of which are concentrated in exons 4 and 10, resulting in aberrant BRG1 expression. We examined the expression of BRG1 in 38 gastric carcinomas and corresponding nonneoplastic mucosa by using the quantitative real-time RT-PCR method. Twenty-three carcinomas (61%) showed increased BRG1 expression in tumor tissue in comparison with that in nonneoplastic mucosa. The T/N ratio (the expression level of BRG1 mRNA in tumor tissues relative to those in corresponding nonneoplastic mucosa) in advanced cases of gastric carcinoma (stages III and IV) was significantly higher than that in cases of stage I and II carcinoma (p = 0.029). Furthermore, gastric carcinomas with lymph node metastasis showed a tendency to express BRG1 at a higher level than gastric carcinomas without metastasis (p = 0.097). We also searched for genetic alterations of the BRG1 gene in 8 gastric carcinoma cell lines and 33 primary gastric carcinomas by PCR-SSCP analysis. No SSCP variants in exons 4, 10 and 16 of the BRG1 gene were found in both gastric carcinoma cell lines and primary gastric carcinomas. These results suggest that, although genetic abnormality of BRG1 might be rare, an increased expression of BRG1 might be associated with the development and progression of gastric carcinoma.

Expression of osteoprotegerin correlates with aggressiveness and poor prognosis of gastric carcinoma.

Osteoprotegerin (OPG), identical with osteoclastogenesis inhibitory factor, is a member of a subgroup of the tumor necrosis factor (TNF)-receptor superfamily, which functions as a soluble decoy receptor. It has been reported that OPG expression is associated with bone metastasis of cancer of the breast and prostate. In the present study, we examined the expression of OPG in gastric carcinomas using immunohistochemistry and reverse-transcription polymerase chain reaction methods, and compared with clinicopathological parameters. The expression of OPG mRNA was confirmed in a gastric carcinoma cell line (MKN-7) and gastric carcinoma tissues. Immunohistochemically, strongly positive staining of OPG was found in 65% (67/103) of gastric carcinomas, whereas OPG protein was not detected in non-neoplastic mucosal epithelia. The expression of OPG protein in gastric carcinoma tissues correlates significantly with depth of tumor invasion, nodal metastases and advanced tumor stage. Furthermore, the prognosis of the cases with strong OPG expression was significantly worse than those with weak or no expression of OPG. These results suggest that OPG may participate in stomach carcinogenesis, invasion and metastasis, and may serve as a novel molecular marker for aggressive gastric cancer.

Loss of heterozygosity and histone hypoacetylation of the PINX1 gene are associated with reduced expression in gastric carcinoma

The expression of PINX1, a possible telomerase inhibitor and a putative tumor suppressor, has not been studied in human cancers, including gastric cancer (GC). We examined expression of PINX1 by quantitative reverse transcription (RT)–PCR in 73 cases of GC, and 45 of these cases were further studied for loss of heterozygosity (LOH) by PCR with microsatellite marker D8S277. Reduced expression (tumor vs normal ratio<0.5) of PINX1 was detected in 50 (68.5%) of 73 cases of GC. GC tissues with reduced expression of PINX1 showed significantly higher telomerase activities as measured by telomeric repeat amplification protocol than those with normal expression of PINX1 (P = 0.031). LOH of PINX1 locus was detected in 15 (33.3%) of 45 cases of GC and was correlated significantly with reduced expression of PINX1 (P=0.031). Expression of PINX1 in a GC cell line, MKN-74, was induced by treatment with trichostatin A (TSA) or nicotinamide (NAM). Chromatin immunoprecipitation assay of MKN-74 cells revealed that acetylation of histone H4 in the 5′ untranslated region (UTR) of PINX1 was enhanced by treatment with TSA or NAM, whereas acetylation of histone H3 was not changed by TSA or NAM. In addition, TSA or NAM treatment led to inhibition of telomerase activity in MKN-74 cells. These results indicate that LOH of PINX1 locus and hypoacetylation of histone H4 in the 5′ UTR of PINX1 are associated with reduced expression of PINX1 in GC.

Small cell carcinoma of the extrahepatic bile duct: Case report and immunohistochemical analysis

A small cell carcinoma of the extrahepatic bile duct in a 75‐year‐old Japanese man is reported. The patient suffered from obstructive jaundice, and percutaneous transhepatic cholangiography‐drainage (PTCD) revealed a massive lesion in the lower common bile duct. Because it was diagnosed as a malignant tumor, pancreaticoduodenectomy was performed. A nodular infiltrating tumor measuring 4.5 × 3.0 × 2.0 cm was located in the intrapancreatic portion of the extrahepatic bile duct. Histologically, the tumor was composed of a dense proliferation of small atypical cells with a little region of high‐grade dysplasia in the adjacent epithelium of the common bile duct. Tumor cells were immunoreactive to neuroendocrine markers such as chromogranin A, synaptophysin, CD56, and Leu7. Although carcinoma cells invaded into pancreas and duodenum, there were no histological findings that indicated the carcinoma arose from the mucosa of either the pancreatic duct or duodenum. These results indicated that the tumor was a small cell carcinoma derived from the epithelium of the extrahepatic bile duct; a rare neoplasm with only a few cases reported. A few neuroendocrine cells were recognized in the adjacent epithelium of the extrahepatic bile duct, suggesting that the tumor cells might be derived from them. Using immunohistochemical examination, no p53 abnormality was found. Tumor cells showed positive nuclear staining for p16, while negative for cyclin D1, suggesting that functional retinoblastoma protein (pRB) might be lost in the p16/pRB pathway, as in small cell lung cancer.

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.