Role of DNA Methylation in the Development of Diffuse-Type Gastric Cancer

Cancer cells exhibit two opposing methylation abnormalities: genome-wide hypomethylation and gene promoter hypermethylation. Downregulation of E-cadherin (CDH1) plays a key role in the development of diffuse-type gastric cancer, and DNA methylation is a major cause of the gene’s silencing. Hereditary diffuse gastric cancer is caused by germline mutation of CDH1 gene, and DNA methylation frequently serves as the second hit completely inactivating the gene. In sporadic diffuse-type gastric cancer, methylation of CDH1 is more prevalent than mutation of the gene. Epstein-Barr virus (EBV)-associated gastric carcinoma (EBV-associated GC) is characterized by concurrent methylation of multiple genes, and diffuse-type gastric cancer is frequently seen among EBV-associated GCs. Patients with pangastritis or enlargedfold gastritis, which are both caused by Helicobacter pylori infection, reportedly have an increased risk for diffuse-type gastric cancer. Notably, the gastric mucosa of enlarged-fold gastritis patients exhibits CDH1 hypermethylation and genome-wide hypomethylation. These data suggest that aberPublished online: January 27, 2011 Yasuhisa Shinomura, MD, PhD First Department of Internal Medicine, Sapporo Medical University S1, W16, Chuo-Ku, Sapporo 064-8543 (Japan) Tel. +81 11 611 2111, Fax +81 11 611 2242 E-Mail shinomura @ sapmed.ac.jp © 2011 S. Karger AG, Basel 0012–2823/11/0834–0241$38.00/0 Accessible online at: www.karger.com/dig D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :3 1: 08 P M Yamamoto /Suzuki /Takamaru / Yamamoto /Toyota /Shinomura Digestion 2011;83:241–249 242 mucosa cells. It is believed that H. pylori infection plays a pivotal role in the development of intestinal-type gastric cancer, which arises from chronic gastritis, atrophy and intestinal metaplasia. Indeed, it is well known that patients with high-grade atrophic gastritis and intestinal metaplasia are at high risk of developing gastric cancer. On the other hand, the sequence of events via which histologically undifferentiated diffuse-type gastric cancers develop is poorly understood, though it is thought that a subset of diffuse-type gastric cancers develop independently of atrophic gastritis or intestinal metaplasia. In contrast to hereditary diffuse gastric cancer (HDGC), H. pylori and/or Epstein-Barr virus (EBV) infections reportedly play essential roles in the development of sporadic diffuse-type gastric cancers. In particular, patients with pangastritis and enlarged-fold gastritis, the cause of which is H. pylori infection, are reportedly at increased risk of developing diffuse-type gastric cancer [2, 4, 5] . Cancer is thought to arise through the accumulation of multiple genetic alterations, leading to activation of oncogenes and loss of function of tumor-suppressor genes. With respect to the latter, mutation of p53 gene is seen in approximately 40% of intestinal-type gastric cancers, but it is rare in diffuse-type gastric cancers [6, 7] . CDH1, which encodes E-cadherin, is frequently mutated in sporadic diffuse-type gastric cancers [8] , and germline mutations of CDH1 are detected in a subset of HDGC patients [9] . In addition, activation of Wnt signaling through mutation of APC is a common feature of colorectal cancer, and APC mutation is also frequently seen in gastric adenoma. By contrast, APC mutation is not often seen in either intestinaland diffuse-type gastric cancers [6, 7, 10] . With respect to oncogenes, activating mutation of CTNNB1, which encodes -catenin, is seen in approximately 20% of intestinal-type gastric cancers [11] , while the frequency of KRAS mutation is low in both histological types [10] . Taken together, these data suggest that, as compared to other cancer types (e.g. colorectal cancers), genetic mutations are relatively infrequent in gastric cancer [12] . A growing body of evidence now suggests that, in addition to genetic alterations, epigenetic changes, including DNA methylation and histone modification, also play important roles in the development and progression of human malignancies [13–16] . Epigenetics are inherited factors that influence gene activity but do not alter primary DNA sequences; among them, DNA methylation is a key event that silences gene expression. It has been hypothesized that DNA methylation initially evolved as a defense mechanism against viruses and other DNA pathogens. Under normal physiological conditions, DNA methylation plays a role in genome imprinting, X-chromosome inactivation and inactivation of repetitive sequences. In cancer, however, two contradicting epigenetic events coexist, namely global hypomethylation, which is mainly observed in repetitive sequences within the genome, and regional hypermethylation, which is frequently associated with CpG islands in gene promoters. Global hypomethylation is thought to be associated with proto-oncogene activation and chromosomal instability, whereas regional hypermethylation leads to inactivation of tumor-suppressor genes. A number of studies provide evidence that both genetic and epigenetic alterations play critical roles in gastric tumorigenesis. For example, approximately 20% of intestinal-type gastric cancers show microsatellite instability that is closely associated with hypermethylation of MLH1 gene [17, 18] . A number of tumor-suppressor and tumor-related genes, including APC, CDH1 (E-cadherin), CHFR, DAPK, GSTP1, p16 and RUNX3, are known to be silenced by hypermethylation in gastric cancer [15, 16, 19] . Moreover, such methylation is frequently observed at premalignant stages of gastric cancer (e.g. with chronic gastritis and intestinal metaplasia), suggesting that aberrant methylation occurs early during the multistep process of gastric carcinogenesis [20–23] . Accumulation of aberrant methylation is thought to promote carcinogenesis through activation of common cancer pathways. For instance, although genetic mutation of APC or CTNNB1 is relatively infrequent in gastric cancer, a number of negative regulators of Wnt signaling, including SFRP1, SFRP2, DKK2, DKK3 and WIF1, are frequently methylated in gastric cancer [24–26] . In addition, methylation of RASSF family genes is thought to serve as an alternative to KRAS mutation in the signaling pathway leading to activation the Ras [27] . Mounting evidence suggests that diffuse-type gastric cancer is strongly associated with aberrant DNA methylation. A subset of cancers that exhibit concurrent hypermethylation of multiple genes is thought to represent a CpG island methylator phenotype (CIMP) [28] . In colorectal cancer, CIMP is strongly associated with MLH1 methylation and microsatellite instability [28] . In gastric cancer, however, CIMP is frequently observed in diffuse-type cancers in which MLH1 methylation and microsatellite instability are less frequent [29] . In this review, we will highlight the contribution made by DNA methylation to the development of diffuse-type gastric cancer, and its clinical application as a potential biomarker. D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :3 1: 08 P M Role of DNA Methylation in Diffuse-Type Gastric Cancer Digestion 2011;83:241–249 243 Hereditary Diffuse Gastric Cancer According to the International Gastric Cancer Linkage Consortium, HDGC is clinically characterized by either (1) two or more documented cases of diffuse gastric cancer in firstor second-degree relatives with at least one diagnosed before age 50 years, or (2) three or more cases of documented diffuse gastric cancer in firstor seconddegree relatives, independent of the age of onset [30] . HDGC is an autosomal-dominant inherent cancer syndrome, and approximately 30% of individuals with a clinical diagnosis of HDGC harbor germline mutation of CDH1 [31, 32] . Among individuals with CDH1 germline mutations, the cumulative risk of advanced gastric cancer by age 80 years is 69% in men and 83% in women [33] . CDH1 is located on chromosome 16q22.1 and encodes E-cadherin, which is a transmembrane homodimeric protein that is central to calcium-dependent adhesion of epithelial cells. The mature protein is comprised of three major domains: a large extracellular domain and smaller transmembrane and cytoplasmic domains. The N-terminal ends of the large extracellular domains of the dimers interact with similar E-cadherin dimers on opposing cell surfaces, while the C-terminal ends of the cytoplasmic domains associate with the actin cytoskeleton via a complex that includes -catenin, -catenin and -catenin. And by competing with APC for binding to  -catenin, E-cadherin also modulates activity in the intracellular catenin signaling pathway [34] . Loss of E-cadherin is believed to lead to loss of cell adhesion, which would promote invasion and metastasis. Among all the reported CDH1 germline mutations, approximately 80% are predicted to generate truncated E-cadherin transcripts (nonsense, splice-site and frameshift mutations), while the remaining 20% are missense mutations [31, 35] . In addition to genetic mutation or allelic loss, epigenetic gene silencing associated with DNA methylation is now recognized as an alternative mechanism by which tumor-suppressor genes are inactivated. Within the mammalian genome, DNA methylation occurs only at cytosine bases located 5 to a guanosine in a CpG dinucleotide ( fig. 1 ). This dinucleotide is actually underrepresented in much of the genome, but short regions ( 1 500 bp in length) known as CpG islands are rich in CpG dinucleotides [36] . The majority of CpG islands are found in the 5 end regions of approximately half of the genes in the human genome, and are generally unmethylated in normal cells. By contrast, a number of tumor-suppressor and tumor-associated genes are hypermethylated and transcriptionally inactivated in cancer cells ( fig. 2 ). CDH1 promoter methylation and intragenic deletions in the wild-type allele are frequently observed in HDGC and CG 5