Toshihiro Tsuneyoshi

PIK3CA mutation and amplification in human lung cancer

To explore the significance of phosphatidylinositol‐3‐kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild‐type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over‐expressed wild‐type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage‐independent growth activity and migration activity of immortalized airway epithelium 16HBE14o– cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild‐type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

Piroxicam and acarbose as chemopreventive agents for spontaneous intestinal adenomas in APC gene 1309 knockout mice

The use of nonsteroidal anti‐inflammatory drugs has been suggested to have a chemopreventive effect against colon carcinoma, through the inhibition of cyclooxygenases 1 and 2, in patients with familial adenomatous polyposis and in animal models. Acarbose, an alpha‐glycosidase inhibitor, may also be chemopreventive. In order to examine the effects of these drugs we employed APC gene knockout mice randomized into 3 groups, one for treatment with piroxicam (0.05% concentration in drinking water), one for acarbose (0.04% concentration in food) and another for the control. After 14 weeks of treatment, mice were killed for quantitation of gastric and intestinal adenomas. Tumor multiplicity in the whole gastrointestinal tract decreased from 33.89±13.07 tumors/mouse in the control group to 17.05±7 tumors/mouse in the piroxicam‐treated group (P<0.001). The decrease in the acarbose‐treated group (29.68±12.86 tumors/mouse) was not significant (P>0.05). The number of tumors ≥3 mm in diameter was also quantified in all gastrointestinal segments. The number of such tumors in the piroxicam group was decreased to 0.56±1.2 tumors/mouse from the control value of 3.78±1.17 tumors/mouse (P<0.001), while in the acarbose‐treated group the number decreased to 2.36±1.7 tumors/mouse (P<0.01). Thus, piroxicam decreases the size and number of gastrointestinal adenomas in APC 1309 knockout mice, while acarbose decreases only the size.

Adenine DNA glycosylase activity of 14 Human MutY homolog (MUTYH) variant proteins found in patients with colorectal polyposis and cancer

Biallelic inactivating germline mutations in the base excision repair MUTYH (MYH) gene have been shown to predispose to MUTYH‐associated polyposis (MAP), which is characterized by multiple colorectal adenomas and carcinomas. In this study, we successfully prepared highly homogeneous human MUTYH type 2 recombinant proteins and compared the DNA glycosylase activity of the wild‐type protein and fourteen variant‐type proteins on adenine mispaired with 8‐hydroxyguanine, an oxidized form of guanine. The adenine DNA glycosylase activity of the p.I195V protein, p.G368D protein, p.M255V protein, and p.Y151C protein was 66.9%, 15.2%, 10.7%, and 4.5%, respectively, of that of the wild‐type protein, and the glycosylase activity of the p.R154H, p.L360P, p.P377L, p.452delE, p.R69X, and p.Q310X proteins as well as of the p.D208N negative control form was extremely severely impaired. The glycosylase activity of the p.V47E, p.R281C, p.A345V, and p.S487F proteins, on the other hand, was almost the same as that of the wild‐type protein. These results should be of great value in accurately diagnosing MAP and in fully understanding the mechanism by which MUTYH repairs DNA in which adenine is mispaired with 8‐hydroxyguanine.

A novel STK11 germline mutation in two siblings with Peutz-Jeghers syndrome complicated by primary gastric cancer.

Patients with Peutz–Jeghers syndrome (PJS) are known to be at risk of gastric cancer (GC), and the STK11 gene is a susceptibility gene for PJS. However, as no cases of PJS with GC in which a STK11 germline mutation has been identified have ever been reported and other susceptibility genes have also been suggested to be involved in PJS, the relation between STK11 germline mutations and GC in PJS is still unknown. In this study, we used sequencing analysis to investigate the STK11, CDH1, and TP53 loci for a germline mutation in two siblings with PJS with primary GC. A novel type of the STK11 germline mutation, c.890delG, encoding a truncated protein (p.Arg297fsX38) was identified, but no germline mutations of the CDH1 and TP53 genes were detected. No inactivation of the wild‐type allele by somatic mutation or chromosomal deletion or hypermethylation at the 5′‐CpG site of STK11 was detected in the GC. This is the first report of a STK11 germline mutation in a PJS patient with GC and should contribute to establishing correlations between the STK11 germline mutations and GC in PJS patients.

Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population

Germline point or small frameshift mutations of the CDH1 (E‐cadherin) gene are known to cause familial gastric cancer (FGC), but the frequency of CDH1 mutations is low in Japanese patients with FGC. Because recent studies have reported germline large genomic deletions of CDH1 in European and Canadian patients with FGC, in the present study we examined DNA samples from 13 Japanese patients with FGC to determine whether similar germline changes were present in CDH1 in this population. Using a sequencing analysis, a 1‐bp deletion (c.1212delC), leading to the production of a truncated protein (p.Asn405IlefsX12), was found in an FGC family; immunohistochemical analysis revealed the loss of CDH1 protein expression in the tumors in this family. Using a combination of multiplex ligation‐dependent probe amplification (MLPA) and RT‐PCR analyses, we also found a large genomic deletion (c.164‐?_387+?del), leading to the loss of exon 3 and the production of a truncated protein (p.Val55GlyfsX38), in another FGC family. The functional effects of the detected mutations were examined using a slow aggregation assay. Significant impairment of cell–cell adhesion was detected in CHO‐K1 cells expressing Ile405fsX12‐ and Gly55fsX38‐type CDH1 compared with cells expressing wild‐type CDH1. Our results suggest that the p.Asn405IlefsX12 and p.Val55GlyfsX38 mutations of the CDH1 gene contribute to carcinogenesis in patients with FGC. This is the first report of CDH1 germline truncating mutations in Japanese patients with FGC. Screening for large germline rearrangements should be included in CDH1 genetic testing for FGC. (Cancer Sci 2011; 102: 1782–1788)

Altered expression of the human base excision repair gene NTH1 in gastric cancer

A base excision repair enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol (Tg), from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against Tg by empty vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced messenger RNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. No associations between clinicopathological factors and NTH1 expression level or localization pattern were detected in the gastric cancers. Next, we found two novel genetic polymorphisms, i.e. c.-163C>G and c.-241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. These findings suggested that downregulation of NTH1 expression and abnormal localization of NTH1 may be involved in the pathogenesis of a subset of gastric cancers.

Association between CDH1 haplotypes and gastric cancer risk in a Japanese population

Objective. A c.−285C > A single nucleotide polymorphism (SNP) in the promoter region of the E-cadherin (CDH1) gene, which is a tumor suppressor in gastric cancer (GC), has been shown to decrease gene transcription, but GC case-control studies of this SNP have yielded controversial results. A haplotype study in an Italian population showed that haplotypes based on three SNPs, including the c.−285C > A, are associated with susceptibility to GC. Hence, the purpose of the present study was to carry out a more comprehensive genetic analysis of CDH1 using haplotype-tagging SNPs (htSNPs) in a Japanese case-control study to identify the CDH1 haplotype associated with susceptibility to GC in a Japanese population. Material and methods. First, 11 SNPs in the CDH1 gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 30 healthy individuals. Haplotype frequencies were estimated with the expectation-maximization algorithm, and 7 common haplotypes of the CDH1 gene whose frequency was at least 3.3% were identified. Next, 5 htSNPs (c.−285C > A, c.48+6T > C, c.164−3159T > C, c.2076C > T, and c.2296−616G > C) were genotyped in a hospital-based case-control study of 148 GC patients and 292 age- and gender-matched healthy controls, and haplotype frequencies based on the 5 htSNPs were estimated. Results. Although none of the 5 htSNPs was related to an overall risk of GC, frequencies of the ATCTG and CTTTG haplotypes were significantly higher and lower, respectively, in the GC cases than in the controls (p<0.05). Conclusions. These results suggest that the ATCTG and CTTTG CDH1 haplotypes may be associated with an increased risk and decreased risk, respectively, of GC in the Japanese population.

OGG1, MYH and MTH1 gene variants identified in gastric cancer patients exhibiting both 8-hydroxy-2′-deoxyguanosine accumulation and low inflammatory cell infiltration in their gastric mucosa

Introduction 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the main DNA modifications produced by reactive oxygen species (ROS). Because 8-OHdG can pair with cytosine and adenine bases during DNA synthesis, when 8-hydroxyguanine (8-OHG) is present in the DNA template, it causes G:C to T:A transversions (Shibutani et al. 1991), and it induces A:T to C:G transversions when 8-hydroxy-dGTP in the nucleotide pool is incorporated into DNA (Maki and Sekiguchi 1992). Because of these transversions, 8-OHdG accumulation is thought to cause carcinogenesis. 8-OHdG accumulation in mammalian cells is prevented by the base excision repair enzymes OGG1, MYH and NEIL1, and by MTH1, an enzyme that removes 8-hydroxy-dGTP from the intracellular nucleotide pool (Nakabeppu 2001). A variety of factors, including sodium chloride, Helicobacter pylori infection and smoking (Tredaniel et al. 1997; Farinati et al. 1998), induce inflammation in the stomach tissue. A considerable inflammatory cell infiltrate in the gastric mucosa causes the production of ROS (Ernst 1999), and ROS are thought to lead to 8-OHdG accumulation in the gastric mucosa (Farinati et al. 1998), suggesting that the level of inflammatory cell infiltration in the stomach may be one of the factors that determine the 8-OHdG level in the stomach. We, therefore, hypothesized that gastric cancer patients with both 8-OHdG accumulation and low level of inflammatory cell infiltration in their stomach have genetic factors that cause them to have a low ability to repair 8-OHdG. We selected 23 patients exhibiting mild or no neutrophil and mono-

Genetic Diagnosis by Polymerase Chain Reaction and Electrospray Ionization Mass Spectrometry: Detection of Five Base Deletion From Blood DNA of a Familial Adenomatous Polyposis Patient

A 5-base deleted mutation of adenomatous polyposis coli (APC) gene was detected by using electrospray ionization mass spectrometry of polymerase chain reaction (PCR) products. Genomic DNA was extracted from a familial adenomatous polyposis patient blood, and a 57-base pairs segment of APC gene was amplified by PCR. The PCR products were purified, digested with restriction endonuclease, purified, and determined by electrospray mass spectrometry.

Identification and characterization of a novel germline p53 mutation in a patient with glioblastoma and colon cancer.

Germline mutations in the p53 tumor suppressor gene have been identified in patients with Li‐Fraumeni syndrome (LFS) and patients with Li‐Fraumeni‐like syndrome (LFL). However, to date, germline p53 mutations in patients not fulfilling the criteria of LFS or LFL have been reported only very rarely. In our study, a novel germline c.584T>C (p.Ile195Thr) mutation of the p53 gene was found in a 21–year‐old male with a glioblastoma and colon cancer. He had no family history of cancer within second‐degree relatives, and loss of the wild‐type p53 allele and overexpression of p53 protein were observed in both tumors. Functional analyses revealed transactivation and growth suppressive function activities of the Thr195‐type p53 to be impaired. These results suggest germline p53 mutations to possibly be responsible for a subset of young adult patient with multiple malignant tumors, even those not meeting the clinical criteria for LFS or LFL.