—  SYMPOSIUM #48  —

Molecular Pathogenesis of Gastrointestinal Neoplasia
Moderators: Dr. Wataru Yasui and Dr. Jeremy Jass

Section 1 - Molecular Pathogenesis of Gastrointestinal Neoplasia: Stomach

Wataru Yasui, Phyu Phyu Aung, Hirofumi Nakayama and Naohide Oue
Department of Molecular Pathology
Hiroshima University


Gastric cancer is one of the most common malignancies worldwide and is the second most frequent cause of cancer-related death. Helicobacter pylori (H. pylori) causes chronic active or atrophic gastritis and intestinal metaplasia. These are believed to be precancerous lesions or predisposing conditions for gastric cancer. Cancer is a chronic proliferative disease with multiple genetic and epigenetic alterations; that is, a disease with altered gene expression. Genetic polymorphism is an important endogenous cause and a fundamental factor for cancer risk. Recent advances in genomic science including global analysis of gene expression have revealed molecular mechanisms of stomach carcinogenesis more in detail. A better knowledge of the molecular basis of gastric cancer may lead to new approach to diagnosis, treatment and prevention. In this symposium, we will describe an overview of molecular pathways of stomach carcinogenesis, mechanisms of epigenetic alterations, importance of genetic polymorphisms, the search for novel genes specific in gastric cancer through serial analysis of gene expression (SAGE), and refer to the clinical implications.

Overview of Molecular Pathway of Stomach Carcinogenesis
A variety of genetic and epigenetic alterations occur during multistep stomach carcinogenesis. Those include activation of oncogenes and growth factors/receptors, inactivation of tumor suppressor genes, DNA repair genes and cell adhesion molecules, abnormalities of cell cycle regulators and so on. Genetic alterations found in gastric carcinoma are gene amplification, point mutation and loss of heterozygosity, while representative epigenetic changes are gene silencing by DNA methylation and overexpression at transcriptional level. Genetic polymorphism predisposes endogenous cause and alters cancer susceptibility. Genetic instability, CpG island methylation, telomerase activation and p53 mutation commonly participate in the early step of stomach carcinogenesis. The amplification and overexpression of the c-met and cyclin E genes are frequently associated with advanced stage. Strong telomerase activity by hTERT expression participates in cellular immortalization in a majority of gastric cancers. Some of these changes occur commonly in both differentiated and undifferentiated types and some differ depending on the histological types or mucin phenotypes (gastric v.s. intestinal). Microsatellite instability is frequently associated with differentiated type gastric cancer of foveolar phenotype with papillary morphology. Precancerous lesions such as intestinal metaplasia and adenoma share alterations similar to those of the differentiated type gastric cancer.

Epigenetic Alterations of Tumor-related Genes
Among various epigenetic alterations, modified gene expression through DNA methylation and chromatin remodeling by histone modification are the most important events. The abnormal methylation of CpG islands associated with tumor suppressor genes can lead to transcriptional silencing, inactivating the gene and participating in tumorigenesis. In gastric cancer, aberrant methylation is involved in the inactivation of various important genes such as p16MTS1/INK4A, CDH1, hMLH1, RAR-beta, RUNX3, MGMT, TSP1, HLTF, RIZ1, and CHFR, with the incidence of methylation raging from 10% to 70%. When analyzed DNA methylation of 12 tumor-related genes, high methylation (5 or more methylated genes) is found more frequently in stage III/IV cancers than in stage I/II cancers, indicating that DNA methylation of tumor-related genes accumulates in conjunction with tumor progression.

Histone modification and chromatin remodeling linked with CpG island methylation play a major role in epigenetic regulation of gene expression. Reduced histone acetylation is significantly associated with depth of tumor invasion and nodal metastasis of gastric cancer . Hypoacetylation of histones H3 and H4 in the p21WAF1/Cip1 promoter region is observed in more than 50% of gastric cancer tissues by chromatin immunoprecipitation, and hypoacetylation of histone H3 in the promoter is associated with reduced expression of p21 regardless of p53 gene status. Up-regulation of histone acetylation by trichostatin A induces growth arrest and apoptosis and suppresses invasion of gastric cancer cells. Therefore, histone acetylation should be a promising target for cancer therapy especially against invasive and metastatic disease.

Genetic Polymorphism and Gastric Carcinoma Risk
Individual variations in cancer risk are associated with genetic polymorphisms that are present in a significant proportion of the normal population. Genetic polymorphism must be crucial in various processes relevant to stomach carcinogenesis including 1) the mucosal protection against H. pylori infection or other carcinogens; 2) the inflammatory response which conditions maintenance, severity and outcome of the H. pylori infection; 3) the functioning of carcinogen detoxification and antioxidant protection; 4) the intrinsic variability of DNA repair processes; and 5) cell proliferation activity. Single nucleotide polymorphism (SNP) (A>G, Ile>Val) is present in the transmembrane domain of the HER-2/c-erbB2. Our case-control study has demonstrated that Val genotype is significantly more frequent in gastric cancer patients than in controls. Gastric cancers of advanced stage are more frequent in patients with Val genotype than those with Ile genotype, suggesting that this SNP could modulate gastric cancer risk and serve as a predictor of risk for a malignant phenotype. Certain genetic polymorphisms are associated with therapeutic efficacy and toxicity of anti-cancer drugs.

Novel Genes of Gastric Cancer Through Serial Analysis of Gene Expression
Serial analysis of gene expression (SAGE) is a powerful technique to allow genome-wide analysis of gene expression in a quantitative manner without a prior knowledge of the sequence of the gene. We have performed on 5 samples of gastric cancer with different histology and stages, and created the largest SAGE libraries of gastric cancer in the world, containing a total of 137,706 expressed tags including unique 38,903 tags (GEO accession number GSE 545). By comparing gene expression profiles between gastric cancer and normal gastric mucosa in combination with quantitative RT-PCR, APOC1, CEACAM6, and YF13H12 were found to be frequently overexpressed in gastric cancer, and FUS, CDH17, COL1A1, COL1A2, and APOE were associated with tumor progression. The immunostaining of CDH17 tended to be associated with intestinal type cancer and the cases with CDH17 expression was significantly more frequent in advanced stage cases than in early stage, and the prognosis of patients with positive CDH17 expression was significantly poorer than that of the negative cases.

To search for gastric cancer-specific genes, we compared gastric cancer SAGE libraries with those of various normal tissues, especially important or crucial organs in the SAGEmap database, and identified 54 candidate genes. Quantitative RT-PCR analysis of these candidates revealed that APIN, TRAG3, CYP2W1, MIA, MMP-10, DKK4, GW112, REGIV, and HORMAD1 were expressed much more highly in gastric cancers than in 14 kinds of normal tissues. Immunostaining for both MIA and MMP-10 was correlated with poor prognosis in advanced gastric cancer. Enzyme-linked immunosorbent assay (ELISA) showed high levels of MMP-10 in most of serum samples (95%) from patients with gastric cancer. In invasion assays, MIA-transfected gastric cancer cells were up to three times more invasive than cells transfected with empty vector. These results suggest that MMP-10 is a good marker for the detection of GC and that MIA and MMP-10 are prognostic factors for gastric cancer. As expression of MIA and MMP-10 is narrowly restricted in cancer, these two molecules may be good therapeutic targets.

REGIV is one of the most up-regulated genes in a SAGE library of a scirrhous-type gastric cancer. Immunostaining of Reg IV is associated with both the intestinal mucin phenotype and neuroendocrine differentiation of gastric cancer. Expression of Reg IV is also detected in colorectal carcinomas and carcinoids and pancreatic cancers, but not in lung and breast cancers. By ELISA, high levels of REGIV are detected in 36% of serum samples from gastric cancer patients with high specificity. In vitro studies using RegIV-transfected cells revealed that RegIV inhibits apoptosis by 5-fluorouracil (5-FU) through induction of EGFR phosphorylation and inhibition of caspase-3 and -9. Expression of REGIV was significantly associated with resistance to the combination chemotherapy of 5-FU and cisplatin. These findings suggest that RegIV may serve as a novel biomarker and an indicator for chemo-resistance of gastric cancer.

To search for novel tumor suppressor genes of gastric cancer, genes with decreased expression in gastric cancer were screened by SAGE data analysis and RT PCR, and CLDN18 (encoding claudin-18) was identified to be down-regulated in more than a half of gastric cancers. By innunostaining, expression of claudin-18 was reduced in several intestinal metaplasia and a majority of gastric adenomas. Down-regulation of claudin-18 was correlated with poor survival in advanced gastric cancers. Down-regulation of claudin-18 was observed in over 70% of gastric cancers with intestinal mucin phenotype. Down-regulation of claudin-18 may be an early event in gastric carcinogenesis and is also a good marker of poor survival in gastric cancer.

Our results of SAGE-based analyses provide a list of candidate genes that are involved in stomach carcinogenesis and may serve as novel diagnostic markers and therapeutic targets of gastric cancer.

References:
  1. Yasui W, et al. : Histone acetylation and gastrointestinal carcinogenesis. Ann NY Acad Sci 983:220-231, 2003

  2. Kuraoka K, Yasui W, et al. : A single nucleotide polymorphism in the transmembrane domain coding region of HER-2 is associated with development and malignant phenotype of gastric cancer. Int J Cancer 107:593-596, 2003

  3. Gonzalez CA , et al. : Genetic susceptibility and gastric cancer risk. Int J Cancer 100:249-260, 2003

  4. Oue N, Yasui W, et al. : Gene expression profile of gastric carcinoma: Identification of genes and tags potentially involved in invasion, metastasis and carcinogenesis by serial analysis of gene expression. Cancer Res 64:2397-2405 2004

  5. Yasui W, et al. : Search for new biomarkers of gastric cancer through serial analysis of gene expression and its clinical implication. Cancer Sci 95:385-392, 2004

  6. Yasui W, et al. : Recent advances in molecular pathobiology of gastric carcinoma. The Diversity of Gastric Carcinoma: Pathogenesis, Diagnosis, and Therapy. (Ed) Kaminishi M, Takubo K, Mafune K, Springer-Verlag, Tokyo , p.51-71, 2005

  7. Oue N, Yasui W, et al. : Expression and localization of RegIV in human neoplastic and non-neoplastic tissues: RegIV expression is associated with intestinal and neuroendocrine differentiation in gastric adenocarcinoma. J Pathol 207:185-198, 2005

  8. Mitani Y, Yasui W, et al. : Histone H3 acetylation is associated with reduced p21WAF1/CIP1 expression in gastric carcinoma. J Pathol 205:65-73, 2005

  9. Sanada Y, Yasui W, et al. : Down-regulation of the claudin-18 gene, identified through serial analysis of gene expression data analysis, in intestinal phenotype of gastric cancer. J Pathol 208:633-642, 2006

  10. Oue N, Yasui W, et al. : Accumulation of DNA methylation is associated with tumor stage in gastric cancer. Cancer 106:1250-1259, 2006

  11. Aung PP, Yasui W, et al. : Systematic search for gastric cancer-specific genes based on SAGE data: melanoma inhibitory activity and matrix metalloproteinase-10 are novel prognostic factors in patients with gastric cancer. Oncogene 25:2546-2557, 2006