Gallbladder cancer (GBC) is relatively rare but has a high mortality rate. One candidate molecule which might be involved in GBC development is prostate stem cell antigen (PSCA), a glycosylphosphatidylinositol-anchored cell surface antigen with a tissue-specific pattern of expression in the epithelium of several organs, such as the prostate, stomach, bladder, and gallbladder. It is up-regulated in a number of cancers including prostate, urinary bladder, and pancreatic cancers, while it is down-regulated in esophageal and gastric cancers, suggesting that PSCA has an oncogenic activity in the former but a tumor suppressor activity in the latter. However, the precise function of PSCA and the regulatory mechanism for its expression in normal and cancer cells are yet to be determined. In this study, immunohistochemical analyses with a specific antibody revealed that PSCA is down-regulated in non-neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis, and cholecystitis (9/17; 53%), and also in adenocarcinoma (40/44; 91%), a common neoplasm in gallbladder. Analyses of the DNA methylation status in the GBC cell lines by bisulfite-Pyrosequencing and a reporter assay for the PSCA promoter activity suggested that the down-regulation is explained, at least partly, by DNA methylation. Moreover, colony formation assay revealed that PSCA has cell-proliferation inhibition activity in the GBC cell lines, which was also observed in vivo. These lines of in vivo and in vitro evidence suggest that PSCA is acting as a tumor suppressor in GBC development.

Gastric cancer (GC) is one of the major malignant diseases worldwide, especially in Asia, where Japan and Korea have the highest incidence in the world. Gastric cancer is classified into intestinal and diffuse types. While the former is almost absolutely caused by Helicobacter pylori infection as the initial insult, the latter seems to include cases in which the role of infection is limited, if any, and a contribution of genetic factors is anticipated. Previously, we performed a genome-wide association study (GWAS) on diffusetype GC by using single nucleotide polymorphisms (SNP) catalogued for Japanese population (JSNP), and identified a prostate stem cell antigen (PSCA) gene encoding a glycosylphosphatidylinositol-anchored cell surface antigen as a GC susceptibility gene. From the second candidate locus identified using the GWAS, 1q22, we found the Mucin 1 (MUC1) gene encoding a cell membranebound mucin protein as another gene related to diffuse-type GC. A two-allele analysis based on risk genotypes of the two genes revealed approximately 95% of Japanese population have at least one of the two risk genotypes, and approximately 56% of the population have both risk genotypes. The two-SNP genotype might offer ample room to further stratify a high GC risk subpopulation in Japan and Asia by adding another genetic and/or non-genetic factor. Recently, a GWAS on the Chinese population disclosed an additional three GC susceptibility loci: 3q13.31, 5p13.1 and 10q23. (Cancer Sci 2013; 104: 1-8) G astric cancer (GC) is one of the major malignant diseases and the second causal of cancer death worldwide.(1) It is usually classified into two types, intestinal and diffuse, a classification which was originally based on histological observation but is recently thought to reflect its pathogenesis.(2) The majority of intestinal-type GC (IGC) arises in the sequence of inflammatory change of the gastric epithelium resulting from bacterial infection; Helicobacter pylori (HP) infectionchronic inflammation -intestinal metaplasia -dysplasia -adenocarcinoma. In contrast, de novo diffuse-type GC (DGC) is thought to emerge in a histologically almost normal epithelium as a consequence of some genetic change that occurred in gastric stem cells and/or epithelial precursor cells, although some cases with DGC might represent dedifferentiated stages of IGC, and a contribution of HP is also suggested.(3) In other words, it is apparent that the pathogenesis of IGC is initiated by HP infection, a class I carcinogen acknowledged by WHO, and therefore IGC is essentially a preventable disease by eradicating HP infection. However, DGC might develop earlier in life than IGC, (4) and no definite DGC-specific environmental risk factor has been established. Therefore, so far we have neither solid strategy nor promising theory to envision a consistent diminution of the incidence of DGC.The incidence of GC has strong geographical and ethnical characteristics. For example, it is one of the rare cancers in North America and Europe, while its incidence is s...

Background:Global hypomethylation has been suggested to cause genomic instability and lead to an increased risk of cancer. We examined the association between the global methylation level of peripheral blood leukocyte DNA and breast cancer among Japanese women.Methods:We conducted a hospital-based case–control study of 384 patients aged 20–74 years with newly diagnosed, histologically confirmed invasive breast cancer, and 384 matched controls from medical checkup examinees in Nagano, Japan. Global methylation levels in leukocyte DNA were measured by LUminometric Methylation Assay. Odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between global hypomethylation and breast cancer were estimated using a logistic regression model.Results:Compared with women in the highest tertile of global methylation level, ORs for the second and lowest tertiles were 1.87 (95% CI=1.20–2.91) and 2.86 (95% CI=1.85–4.44), respectively. Global methylation levels were significantly lower in cases than controls, regardless of the hormone receptor status of the cancer (all P values for trend <0.05).Interpretation:These findings suggest that the global methylation level of peripheral blood leukocyte DNA is low in patients with breast cancer and may be a potential biomarker for breast cancer risk.

The power of an SNP‐based genome‐wide association study (GWAS) was first demonstrated in Japan using the JSNP database and is currently a major strategy adopted around the world for a number of common diseases including cancers. The hypothesis‐free strategy can lead us to a novel hypothesis for carcinogenesis and may contribute to identifying a high risk group for research and, in the future, practice of personalized prevention. We performed a GWAS on diffuse‐type gastric cancer and identified a significant association with SNPs in the PSCA (prostate stem cell antigen) gene. The association was validated by a Korean gastric case‐control analysis. The PSCA protein is expressed predominantly in the stem cell/precursor‐rich region of the gastric epithelium, which is considered as the origin of diffuse‐type gastric cancer, and showed tumor suppressor‐like characteristics. Individuals with a low PSCA promoter activity are susceptible to diffuse‐type gastric cancer. By contrast, the polymorphism does not significantly predispose to intestinal‐type gastric cancer, congruous to the hypothesis of the two distinct carcinogenesis pathways for the two major types of gastric cancer. In addition to publication on a specific gene, the sharing of GWAS data through a database on the web is expected to accelerate validation and discovery by other investigators. GeMDBJ (Genome Medicine Database of Japan), started in 2005 in Japan, is one of such attempts. Moreover, the advent of “next generation” sequencers may herald a new era in which the poorly explored domains of the genetic architecture of disease susceptibility may be unveiled. (Cancer Sci 2010)

Global hypomethylation of leukocyte DNA has been associated with an increased risk of cancer. As dietary and genetic factors related to one-carbon metabolism may influence both the methylation and synthesis of DNA, we investigated associations between these factors and the global methylation level of peripheral blood leukocyte DNA based on a cross-sectional study of 384 Japanese women. Dietary intake of folate and vitamins B2, B6, and B12 was assessed with a validated semiquantitative food frequency questionnaire. Five polymorphisms in methylenetetrahydrofolate reductase (MTHFR) (rs1801133 and rs1801131), methionine synthase (MTR) (rs1805087), and methionine synthase reductase (MTRR) (rs10380 and rs162049) were genotyped. Global DNA methylation of leukocyte DNA was quantified using Luminometric Methylation Assay. A linear trend of association between methylation and dietary and genetic factors was evaluated by regression coefficients in a multivariable linear regression model. Mean global methylation level (standard deviation) was 70.2% (3.4) and range was from 59.0% to 81.2%. Global methylation level significantly decreased by 0.36% (95% confidence interval, 0.03-0.69) per quartile category for folate level. Subgroup analysis suggested that alcohol drinking modified the association between folate intake and global methylation level (P interaction = 0.01). However, no statistically significant association was observed for intake of vitamins B2, B6, and B12, alcohol consumption, or five single nucleotide polymorphisms of MTHFR, MTR, and MTRR. We found that higher folate intake was significantly associated with a lower level of global methylation of leukocyte DNA in a group of healthy Japanese females. (Cancer Sci 2012; 103: 2159-2164 D NA methylation plays an important role in the epigenetic mechanism of gene regulation (1,2) and cellular differentiation.(3) Aberrant genomic DNA methylation, both in specific genes and in the genome overall, is widely recognized to be associated with cancer.(4) For example, hypermethylation at promoter CpG islands in tumor suppressor genes is an important means of silencing transcription in carcinogenesis.(4) Global DNA hypomethylation in normally methylated regions is thought to contribute to carcinogenesis through the induction of genomic instability.(4) In addition, some previous evidence suggests that DNA hypomethylation could lead to the activation of oncogenes, and global DNA hypomethylation has been linked to hypomethylation in multiple promoter CpG islands.(4,5) Although many studies have investigated aberrant DNA methylation at the tissue level, there is great interest in epigenetic markers in peripheral blood and several epidemiological studies have found that hypomethylation of global peripheral blood cell DNA is associated with an increased cancer risk.(6-9) However, determinants of global methylation level among healthy individuals remain largely unexplored.Folate and vitamin Bs in one-carbon metabolism are cofactors and cosubstrates for methylation and nucleic acid ...

Previous genomewide association studies identified prostate stem cell antigen (PSCA) as a gastric cancer (GC) susceptibility gene and showed an association between GC and the T allele of the single nucleotide polymorphism rs2294008 (C/T) in this gene. The protein product of this gene inhibits cell growth, and the T allele significantly suppresses the transcriptional activity of the À3.2 kb PSCA upstream region. However, the mechanism remains unknown. In this study, we conducted reporter assays using the PSCA upstream region containing the C allele and identified the region from À200 to +38 bp of the transcription initiation site of the gene as a critical region of the À3.2 kb PSCA upstream region. We found that introducing the T allele at rs2294008 generated a consensus binding sequence for the Polycomb group transcription factor Yin Yang 1 (YY1) and that disruption of the consensus sequence restored the transcriptional activity to the À3.2 kb PSCA upstream region. These findings imply that the T allele significantly suppresses PSCA expression in vivo by recruiting YY1 to its promoter, which eventually predisposes gastric epithelial cells to GC development.

Background: Although global hypomethylation of leukocyte DNA has been associated with an increased risk of several sites of cancer, including breast cancer, determinants of global methylation level among healthy individuals remain largely unexplored. Here, we examined whether postmenopausal endogenous sex hormones were associated with the global methylation level of leukocyte DNA. Methods: A cross-sectional study was conducted using the control group of a breast cancer case-control study in Nagano, Japan. Subjects were postmenopausal women aged 55 years or over who provided blood samples. We measured global methylation level of peripheral blood leukocyte DNA by luminometric methylation assay; estradiol, estrone, androstenedione, dehydroepiandrosterone sulfate, testosterone and free testosterone by radioimmunoassay; bioavailable estradiol by the ammonium sulfate precipitation method; and sex-hormone binding globulin by immunoradiometric assay. A linear trend of association between methylation and hormone levels was evaluated by regression coefficients in a multivariable liner regression model. A total of 185 women were included in the analyses. Results: Mean global methylation level (standard deviation) was 70.3% (3.1) and range was from 60.3% to 79.2%. Global methylation level decreased 0.27% per quartile category for estradiol and 0.39% per quartile category for estrone while it increased 0.41% per quartile category for bioavailable estradiol. However, we found no statistically significant association of any sex hormone level measured in the present study with global methylation level of leukocyte DNA. Conclusions: Our findings suggest that endogenous sex hormones are not major determinants of the global methylation level of leukocyte DNA.

Abstract. Prostate stem cell antigen (PSCA) is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein and exhibits an organ-dependent expression pattern in cancer. PSCA is upregulated in prostate cancer and downregulated in gastric cancer. PSCA is expressed in a variety of human organs. Although certain studies previously demonstrated its expression in the mammalian and avian brain, its expression in the human brain has not been thoroughly elucidated. Additionally, it was previously reported that PSCA is weakly expressed in the astrocytes of the normal human brain but aberrantly expressed in glioma, suggesting that PSCA is a promising target of glioma therapy and prostate cancer therapy. The current study identified PSCA expression in the neural and choroid plexus cells of the normal human brain by immunohistochemistry. In brain tumors, PSCA was expressed in medulloblastoma and glioma, and its expression was also observed in papilloma and papillary carcinoma of the choroid plexus, ependymoma and meningioma. The results suggest that PSCA may have a tumor-promoting function in brain tumors and is a potential target for their therapy. However, its expression in normal neuronal and choroid plexus cells implies that a PSCA-targeted therapy may lead to certain adverse phenomena.