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Papers - KANAI Yae

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Expression status of RUNX1/AML1 in normal gastric epithelium and its mutational analysis in microdissected gastric cancer cells

Usui, Takebumi, Aoyagi, Kazuhiko, Saeki, Norihisa, Nakanishi, Yukihiro, Kanai, Yae, Ohki, Misao, Ogawa, Kenji, Yoshida, Teruhiko, Sasaki, Hiroki

INTERNATIONAL JOURNAL OF ONCOLOGY (PROFESSOR D A SPANDIDOS) 29 ( 4 ) 779 - 784 2006.10

Research paper (scientific journal), ISSN 1019-6439

Although Runt-related transcription factors RUNXs (RUNX1-3) have a high similarity in their structure, only RUNX3 is known to be involved in gastric carcinogenesis. First, we examined mRNA expression of these three RUNX genes in the gastric mucosa, and, finding only RUNX2 was not expressed there, we further investigated RUNX1 and RUNX3 expression in three regions including the pit, isthmus/ neck, and gland regions of the human normal stomach and whether RUNX1 is involved in gastric carcinogenesis. The mRNA expression of RUNX1 and RUAX3 was examined by use of the three regions isolated by laser-captured microdissection (LCM) and by use of primary gastric cancer tissues. Furthermore, RUNX1 mutational analysis was performed in the cancer cells, which also were isolated from 44 paraffin-embedded gastric cancer tissues by LCM. RUNX1 was coexpressed with RUNX3 in the pit region, and has cell growth-inhibition activity similar to RUNX3. RUNX3 has been reported to be suppressed by DNA methylation in a subset of gastric cancers; however, the expression of RUNX1 mRNA was observed in all of the gastric cancer cell lines and gastric cancer tissues that we examined. No RUNX1 mutation was found in the 44 gastric cancer patients. Although RUNX1 is similar to RUNX3 in both the expression pattern in the stomach and its cell growth-inhibition activity, RUNX1 is not involved in most cases of gastric cancers. These results suggest that the transcriptional target genes are different between these two family genes.

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DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas

Peng, Dun-Fa, Kanai, Yae, Sawada, Morio, Ushijima, Saori, Hiraoka, Nobuyoshi, Kitazawa, Sohei, Hirohashi, Setsuo

CARCINOGENESIS (OXFORD UNIV PRESS) 27 ( 6 ) 1160 - 1168 2006.06

Research paper (scientific journal), ISSN 0143-3334

To evaluate the significance of alterations in DNA methylation during multistage carcinogenesis of the pancreas, tissue samples of 13 peripheral pancreatic duct epithelia showing no remarkable histological changes without inflammatory background (DE), 20 peripheral pancreatic duct epithelia showing no remarkable histological changes with inflammatory background (DEI), 40 pancreatic intraepithelial neoplasias (PanIN) and 147 areas of ductal carcinoma were microdissected from surgically resected specimens from 58 patients and were embedded into agarose beads. The embedded tissue samples were subjected to methylation-specific PCR (MSP) to evaluate the DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTPI, TIMP-3, CDH1 and DAPK-1 genes. The prevalence of DNA methylation of at least one of the 12 genes and the average number of methylated genes were significantly higher in both DEI (60% and 0.85 +/- 0.88, P = 0.0151 and P = 0.0224, respectively) and PanIN (67.5% and 0.95 +/- 0.85, P = 0.0014 and P = 0.0028, respectively) than in DE (15.4% and 0.15 +/- 0.38), and were further increased in ductal carcinoma (98.3% and 2.50 +/- 1.35, P < 0.0001 and P < 0.0001, respectively). The BRCA1, APC, p16 and TIMP-3 genes were frequently methylated in ductal carcinoma (60.3, 58.6, 39.3 and 30.9%, respectively). Considerable heterogeneity of DNA methylation status was observed among multiple microdissected areas from individual ductal carcinomas, and the number of methylated genes per area was significantly correlated with poorer tumor differentiation (P = 0.0249). The average number of methylated genes in ductal carcinomas was significantly correlated with DNMT1 protein expression level (P = 0.0093). These data suggest that accumulation of DNA methylation of multiple tumor-related genes is involved in multistage carcinogenesis of the pancreas from early precancerous stages to malignant progression and that DNMT1 protein overexpression may be responsible for this aberrant DNA methylation.

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Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissues.

Arai E, Kanai Y, Ushijima S, Fujimoto H, Mukai K, Hirohashi S

Int J Cancer (WILEY-LISS) 119 ( 2 ) 288 - 296 2006

Accepted, ISSN 00207136

To evaluate the significance of altered DNA methylation during renal tumorigenesis, tumorous tissues (T) and corresponding nontumorous renal tissues (N) from 94 patients with renal tumors, and normal renal tissues (C) from 16 patients without renal tumors were investigated. DNA methylation status on CpG islands of the p16, human MutL homologue 1 (hMLHI), von-Hippel Lindau (VHL) and thrombospondin-I (THBS-1) genes and the methylated in tumor (MINT) -1, -2, -12, -25 and -31 clones and DNA methyltransferase (DNMT) 1 expression were examined by bisulfite modification and immunohistochemistry, respectively. The average number of methylated CpG islands was significantly higher in N than in C, and was even higher in T. The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding conventional renal cell carcinomas (RCCs). The average number of methylated CpG islands in RCCs was significantly correlated with macroscopic configuration with extratiodular or multinodular growth, higher histological grade, infiltrating growth pattern and vascular involvement. The recurrence-free survival rate of patients with RCCs showing accumulation of DNA methylation was significantly lower than that of patients not showing this feature. The incidence of nuclear immunoreactivity for DNMT1 tended to be higher in proximal tubules from N than in those from C, and was significantly higher in RCCs. From the viewpoint of altered DNA methylation, N is at the precancerous stage, and N showing accumulation of DNA methylation may generate more malignant RCCs. Regional DNA hypermethylation may be associated with renal tumorigenesis from a precancerous condition to malignant progression and become a predictor of patient prognosis. (c) 2006 Wiley-Liss, Inc.

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Leiomyosarcoma of the renal vein: arterial encasement on contrast-enhanced dynamic computed tomography.

Maeda T, Tateishi U, Fujimoto H, Kanai Y, Sugimura K, Arai Y

Int J Urol (BLACKWELL PUBLISHING) 13 ( 5 ) 611 - 612 2006

Accepted, ISSN 09198172

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Solid-pseudopapillary neoplasms of the pancreas in men and women: do they differ?

Takahashi Y, Hiraoka N, Onozato K, Shibata T, Kosuge T, Nimura Y, Kanai Y, Hirohashi S

Virchows Arch (SPRINGER) 448 ( 5 ) 561 - 569 2006

Accepted, ISSN 09456317

Solid-pseudopapillary neoplasms (SPNs) of the pancreas are uncommon and occur preferentially in young women. The question whether the features of SPNs occurring in men differ from those in women has not yet been studied. For a better understanding of the clinicopathological features of SPNs of both sexes, we studied a series of 14 tumors surgically resected at a Japanese hospital within a period of 14 years. This series was composed of seven men and seven women. All these SPNs demonstrated nuclear and cytoplasmic accumulation of beta-catenin protein in immunohistochemistry and 86% of them had activating mutations of beta-catenin gene. No pancreatic neuroendocrine tumors showed such immunohistochemical findings and genetic alterations. In our series, most SPNs in women showed encapsulation by thick fibrous tissue and massive degenerative changes. Most SPNs in men exhibited solid components without prominent degenerative changes, even though they were of a similar size to those in women. These findings suggest that SPNs in men tend to be a solid mass with slower progression of degenerative changes during their growth compared to that in women. Nuclear accumulation of beta-catenin appears to be a useful marker of SPN, which allows male SPNs to be correctly diagnosed despite their less typical features.

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Epigenetic instability and chromosomal instability in hepatocellular carcinoma.

Katoh H, Shibata T, Kokubu A, Ojima H, Fukayama M, Kanai Y, Hirohashi S

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 168 ( 4 ) 1375 - 1384 2006

Accepted, ISSN 00029440

The aim of this study was to clarify the association between the epigenetic instability phenotype and the chromosomal instability phenotype in primary hepatocellular carcinoma (HCC). Sixty primary HCC tumors were examined. Methylation status for nine CpG islands (the p16, COX2, GSTP1, RASSF1A, E-cadherin, and APC gene promoters, and the MINT 1, 25, and 31 clones) was evaluated by methylation-specific polymerase chain reaction. Chromosomal structural alterations of these 60 HCC tumors were characterized in our previous study by using whole genomic array-based comparative genomic hybridization. We found that the epigenetic instability phenotype and the chromosomal instability phenotype are not mutually exclusive in hepatocarcinogenesis and that they do not show a simple cause-and-effect relationship. Hepatitis virus infection in the background liver was significantly associated with these instability phenotypes. Furthermore, we identified an epigenetic instability-dependent HCC that shows frequent epigenetic aberrations without chromosomal instability. it was noteworthy that epigenetic instability-positive and -negative HCCs displayed distinctive combinations of chromosomal structural alterations. In summary, by combined analyses of genetic and epigenetic aberration profiles in HCC, we obtained a comprehensive view of genomic alterations in hepatocarcinogenesis. Our results have clinical relevance because epigenetic instability-dependent HCCs may respond well to methylation inhibitory therapies.

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Promoter hypermethylation of the potential tumor suppressor DAL-1/4.1B gene in renal clear cell carcinoma

Yamada D, Kikuchi S, Williams YN, Sakurai-Yageta M, Masuda M, Maruyama T, Tomita K, Gutmann DH, Kakizoe T, Kitamura T, Kanai Y, Murakami Y

Int J Cancer (WILEY-LISS) 118 ( 4 ) 916 - 923 2006

Accepted, ISSN 00207136

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed. in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convuluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%). surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC. (c) 2005 Wiley-Liss, Inc.

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21 Role of Immunohistochemical Expression of DMA Methyltransferase 1 Protein in Gastric Carcinoma

Kanai Y., Arai E., Etoh T.

Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas (Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas) 4 257 - 261 2006

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DNA hypomethylation on pericentromeric satellite regions significantly correlates with loss of heterozygosity on chromosome 9 in urothelial carcinomas.

Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 173 ( 1 ) 243 - 246 2005

Accepted, ISSN 00225347

Purpose: DNA methylation has important roles in genomic stability. Accordingly DNA hypomethylation on pericentromeric satellite regions may induce chromosomal instability through heterochromatin decondensation and chromosomal recombination enhancement. We elucidated the significance of aberrant DNA methylation on pericentromeric satellite regions during urothelial carcinogenesis.
Materials and Methods: We examined DNA methylation status on satellites 2 and 3 by Southern blotting and determined the allelic status of chromosome 9 using 6 microsatellite markers (D9S775, D9S925, D9S304, D9S303, D9S283 and D9S747) in 27 transitional cell carcinomas of the bladder, ureter or renal pelvis and corresponding noncancerous tissues.
Results: DNA hypomethylation on satellites 2 and 3 was detected in 2 (7%) and no (0%) noncancerous tissues, and in 11 (41%) and 12 (44%) urothelial carcinomas, respectively. DNA hypomethylation in urothelial carcinomas significantly correlated with histological grade (p = 0.0012 and 0.0043), invasion depth (p = 0.0055 and 0.0228) and morphological structure (papillary vs nodular, p = 0.0161 and 0.0297) for satellites 2 and 3, respectively. Loss of heterozygosity on at least 1 locus of chromosome 9 was detected in 14 urothelial carcinomas (52%). DNA hypomethylation on satellites 2 (p = 0.0098) and 3 (p = 0.0034) significantly correlated with loss of heterozygosity on chromosome 9.
Conclusions: DNA hypomethylation on pericentromeric satellite regions may participate in the development and progression of urothelial carcinomas by inducing loss of heterozygosity on chromosome 9.

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Clinicopathologic significance of dysadherin expression in cutaneous malignant melanoma: immunohistochemical analysis of 115 patients.

Nishizawa A, Nakanishi Y, Yoshimura K, Sasajima Y, Yamazaki N, Yamamoto A, Hanada K, Kanai Y, Hirohashi S.

Cancer (JOHN WILEY & SONS INC) 103 ( 8 ) 1693 - 1700 2005

Accepted, ISSN 0008543X

BACKGROUND. The E-cadherin-mediated cell adhesion system is frequeutly inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently, the authors reported a novel cell membrane glycoprotein, dysadherin, which has an anti-cell-cell adhesion function and down-regulates E-cadherin.
METHODS. Expression of both dysadherin and E-cadherin was investigated immunohistochemically in 115 patients with cutaneous malignant melanoma to determine the correlation between the 2 molecules and their associations with both patient survival and the clinicopathologic features of the tumors.
RESULTS. Dysadherin and E-cadherin were expressed at the cell membranes of melanoma cells. Fifty-two percent of the tumors showed dysadherin immunopositivity, and 91% of the tumors showed reduced E-cadherin immunopositivity. There was no significant inverse correlation between dysadherin expression and E-cadherin expression. Increased dysadherin expression was significantly correlated with nodular subtype (P = 0.042), Clark level (P < 0.001), tumor thickness (P < 0.001), ulceration (P = 0.008), lymph node metastasis (P < 0.001), high TNM classification (P < 0.001), and poor patient survival (P < 0.001). Multivariate analysis of patient survival revealed that increased dysadherin expression was a significant predictor of poor Survival (P < 0.001).
CONCLUSIONS. Thus, increased expression of dysadherin was a significant indicator of poor prognosis in patients with cutaneous malignant melanoma. (c) 2005 American Cancer Society.

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Array-based comparative genomic hybridization analysis of high-grade neuroendocrine tumors of the lung.

Peng WX, Shibata T, Katoh H, Kokubu A, Matsuno Y, Asamura H, Tsuchiya R, Kanai Y, Hosoda F, Sakiyama T, Ohki M, Imoto I, Inazawa J, Hirohashi S.

Cancer Sci (BLACKWELL PUBLISHING) 96 ( 10 ) 661 - 667 2005

Accepted, ISSN 13479032

We examined a large number of primary high-grade neuroendocrine tumors of the lung (10 small cell lung carcinomas and 31 large cell neuroendocrine carcinomas) by using array-based comparative genomic hybridization using microarrays spotted with 800 bacterial artificial chromosome clones containing tumor-related genes from throughout the human genome. We identified the genome-wide copy number alteration profiles of these tumors, including recurrent amplifications located at 2q21.2, 3q21-27, 3q26, 3q27-29, 5p14.2, 5p13, 7q21.1, 8q21, and 8q24 and homozygous deletions at 1p36, 4p16, 4p16.3, 9p21.3, 9p21, 19p13.3, and 20q13. Our results revealed that small cell lung carcinomas and large cell neuroendocrine carcinomas have multiple characteristic chromosomal alterations in common, but that distinctive alterations also exist between the two subtypes. Moreover, we found that the two subtypes undergo different processes of accumulating these genetic alterations during tumor development. By comparing the genetic profiles with the clinicopathological features, we discovered many chromosomal loci whose alterations were significantly associated with clinical stage and patient prognosis. These results will be valuable for evaluating clinical status, including patient prognosis, and for identifying novel molecular targets for effective therapies.

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Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.

Yoshida Y, Shibata T, Kokubu A, Tsuta K, Matsuno Y, Kanai Y, Asamura H, Tsuchiya R, Hirohashi S.

Lung Cancer (ELSEVIER IRELAND LTD) 50 ( 1 ) 1 - 8 2005

Accepted, ISSN 01695002

A hypothesis of multistep carcinogenesis of lung adenocarcinoma from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma through bronchioloalveolar carcinoma (BAC) has been proposed. However, the genetic alterations that play a role during these processes are not yet clear. Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in lung adenocarcinoma. We examined the status of EGFR mutations in AAH and BAC to elucidate the role they play during multistage of lung adenocarcinoma. We found somatic EGFR mutations in 3% (1/35) of AAH, 10.8% (4/37) of BAC and 41.9% (13/31) of invasive adenocarcinoma. Sixteen of 18 EGFR mutations were found in exon 19 and two were in exon 21. Among the 16 EGFR mutations in exon 19, 13 were deletions of 15 bp and one was an insertion/duplication of 18 bp. Mutations of the K-ras gene were detected in 26.7% (8/30) of AAH, 16.7% (5/30) of BAC and 10% (3/30) of invasive adenocarcinoma. None of the tumors with EGFR mutations had K-ras mutation simultaneously. Patients who had invasive adenocarcinoma with EGFR mutations were younger than those without mutations (60.6 versus 67.4 years, p = 0.03). These results suggest that tumors with EGFR mutations may progress more rapidly and develop into invasive cancer faster than those without mutations. Alternatively it is also possible that some invasive adenocarcinomas with EGFR mutations may not follow the AAH-adenocarcinoma sequence. We analyzed 24 patients with multiple lung lesions and 13 patients had at least one lesion that had either an EGFR or K-ras mutation. In all cases each lesion had a different mutation status. This finding suggests that the genetic alterations responsible for the development of lung adenocarcinoma occur randomly even under exposure to the same carcinogen. © 2005 Elsevier Ireland Ltd. All rights reserved.

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Genetic classification of lung adenocarcinoma based on array-based comparative genomic hybridization analysis: its association with clinicopathologic features.

Shibata T, Uryu S, Kokubu A, Hosoda F, Ohki M, Sakiyama T, Matsuno Y, Tsuchiya R, Kanai Y, Kondo T, Imoto I, Inazawa J, Hirohashi S.

Clin Cancer Res (AMER ASSOC CANCER RESEARCH) 11 ( 17 ) 6177 - 6185 2005

Accepted, ISSN 10780432

The array-based comparative genomic hybridization using microarrayed bacterial artificial chromosome clones allows high-resolution analysis of genome-wide copy number changes in tumors. To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, we used laser-capture microdissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. We identified a large number of chromosomal numerical alterations, including frequent amplifications on 7p12, 11q13,12q14-15, and 17q21, and two homozygous deletions on 9p21 and one on 8p23. Unsupervised hierarchical clustering analysis of multiple alterations revealed three subgroups of lung adenocarcinoma that were characterized by the accumulation of distinct genetic alterations and associated with smoking history and gender. The mutation status of the epidermal growth factor receptor (EGFR) gene was significantly associated with specific genetic alterations and supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival. Our results suggest that there exist multiple molecular carcinogenesis pathways in lung adenocarcinoma that may associate with smoking habits and gender, and that genetic cancer profiling will reveal previously uncharacterized genetic heterogeneity of cancer and be beneficial in estimating patient prognosis and discovering novel cancer-related genes including therapeutic targets.

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Prognostic significance of tissue factor in pancreatic ductal adenocarcinoma.

Nitori N, Ino Y, Nakanishi Y, Yamada T, Honda K, Yanagihara K, Kosuge T, Kanai Y, Kitajima M, Hirohashi S.

Clin Cancer Res (AMER ASSOC CANCER RESEARCH) 11 ( 7 ) 2531 - 2539 2005

Accepted, ISSN 10780432

Tissue factor (TF) is a transmembrane glycoprotein that plays roles in the blood coagulation and intracellular signaling pathways, and has also been suggested to modulate the biological behavior of cancer cells. In order to examine the clinicopathologic significance of TF expression in pancreatic ductal adenocarcinoma, TF expression was determined by immunohistochemistry using a newly raised anti-TF monoclonal antibody in 113 patients who had undergone surgical resection of pancreatic ductal adenocarcinoma. According to the incidence of tumor cell immunopositivity, patients were divided into "negative TF (0%), "weak TF (<25%), or "high TF (25% or more) groups, which accounted for 11.6% (n = 13), 44.2% (n = 50), and 44.2% (n = 50) of the total, respectively. Increased TF expression was correlated with the extent of the primary tumor (P = 0.0043), lymph node metastasis (P = 0,0043), lymphatic distant metastasis (P 0.0039), advanced tumor-node-metastasis stage (P = 0.0002),and high tumor grade (P 0,0164). Multivariate analysis using the Cox proportional hazards model showed that high TF expression was an independent negative predictor for survival (hazard ratio, 2.014; P = 0.0076). Moreover, patients with TF-negative tumors had a significantly better prognosis even if lymph node metastasis was present (P < 0.0001). We also showed that TF knockdown by RNA interference suppressed the invasiveness of a pancreatic adenocarcinoma cell line in vitro. These results indicate that TF expression may contribute to the aggressiveness of pancreatic ductal adenocarcinoma by stimulating tumor invasiveness, and that evaluation of the primary tumor for TF expression may identify patients with a poor prognosis.

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Genetic profile of hepatocellular carcinoma revealed by array-based comparative genomic hybridization: identification of genetic indicators to predict patient outcome.

Katoh H, Shibata T, Kokubu A, Ojima H, Loukopoulos P, Kanai Y, Kosuge T, Fukayama M, Kondo T, Sakamoto M, Hosoda F, Ohki M, Imoto I, Inazawa J, Hirohashi S.

J Hepatol (ELSEVIER SCIENCE BV) 43 ( 5 ) 863 - 874 2005

ISSN 01688278

Background/Aims: We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma.
Methods: We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones.
Results: Frequent (> 30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2,2q34,3q11.2,5p14.2,5q13.2-14,7p22,7p14. 2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (> 5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators.
Conclusions: Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Loss of blood group A antigen expression in bladder cancer caused by allelic loss and/or methylation of the ABO gene.

Chihara Y, Sugano K, Kobayashi A, Kanai Y, Yamamoto H, Nakazono M, Fujimoto H, Kakizoe T, Fujimoto K, Hirohashi S, Hirao Y.

Lab Invest (NATURE PUBLISHING GROUP) 85 ( 7 ) 895 - 907 2005

Accepted, ISSN 00236837

Loss of ABO blood group antigen expression has been reported in transitional cell carcinoma ( TCC) of the bladder. Synthesis of the ABO blood group antigen was genetically determined by allelic variants of the ABO gene assigned on 9q34.1. We analyzed loss of heterozygosity ( LOH) and promoter hypermethylation of the ABO gene in TCC and compared them with alterations of A antigen expression in TCC, dysplasia and normal urothelium. A total of 81 samples of TCC of the bladder obtained from transurethral resection ( TUR) ( n = 44) and radical cystectomy ( n = 37) were examined. Expression of the A antigen was evaluated by immunohistochemical staining ( IHC) using anti-A antigen monoclonal antibody. LOH of the ABO gene locus was examined by blunt-end single-strand DNA conformational polymorphism ( SSCP) analysis using flouresence-based auto sequencer. Promoter hypermethylation of the ABO gene were examined by bisulfite PCR-SSCP ( BiPS) analysis and/or methylation-specific PCR ( MSP). Loss of A allele and/or hypermethylation were significantly associated with abnormal expression of the A antigen in cases undergoing TUR ( P = 0.02) and radical cystectomy ( P = 0.0005). For the analysis of the concomitant dysplasia in 23 cases with TCC of the bladder, the expression of the A antigen was maintained, regardless of the A allelic loss or methylation status in the tumor. In conclusion, A allelic loss and hypermethylation in the promoter region of the ABO gene showed significant correlation with reduction of A antigen expression in TCC, while the expression of the A antigen is maintained in concomitant dysplasia or normal urothelium, suggesting that loss of the ABO gene and/or its promoter hypermethylation is a specific marker for TCC.

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Increased DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas.

Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kosuge T, Hirohashi S.

Cancer Sci (BLACKWELL PUBLISHING) 96 ( 7 ) 403 - 408 2005

Accepted, ISSN 13479032

Aberrant DNA methylation has been shown to play an important role during multistage carcinogenesis in various human organs. The aim of the present study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during pancreatic carcinogenesis. Immunohistochemical analysis of DNMT1 in 48 peripheral pancreatic duct epithelia showing no remarkable histological findings without an inflammatory background (DE), 54 peripheral pancreatic duct epithelia with an inflammatory background (DEI), 188 pancreatic intraepithelial neoplasias (PanIN), and 220 areas of invasive ductal carcinoma from surgical specimens resected from 100 patients, was carried out. The average incidence of DNMT1 immunoreactivity increased progressively from DE to DEI (P = 0.003), from DE and DEI to PanIN (P < 0.0001), among PanIN with different grades of dysplasia (from PanIN I to PanIN II, P = 0.0012), from PanIN to invasive ductal carcinomas (P < 0.0001) and among invasive ductal carcinomas with different grades of histological differentiation (from well or moderately to poorly differentiated adenocarcinomas, P < 0.0001). High-level DNMT1 protein expression in invasive ductal carcinomas was correlated significantly with an advanced t category (P=0.0224) and an advanced stage (P=0.0294). Moreover, patients with invasive ductal carcinomas showing high-level DNMT1 protein expression had a poorer outcome (P = 0.0469). These data suggest that increased DNMT1 protein expression participates in multistage pancreatic carcinogenesis from the precancerous stage to malignant progression of ductal carcinomas and may be a biological predictor of poor prognosis.

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DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis.

Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 173 ( 5 ) 1767 - 1771 2005

Accepted, ISSN 00225347

Purpose: We elucidated the significance of aberrant DNA methylation on multiple CpG islands and its correlation with DNA methyltransferase DNMT1 protein expression during urothelial carcinogenesis.
Materials and Methods: We examined the DNA methylation status on multiple CpG islands by methylation specific polymerase chain reaction and combined bisulfite restriction enzyme analysis in 12 specimens of normal urothelium, 23 of noncancerous urothelium showing no remarkable histological changes obtained from patients with bladder cancer (NBC) and 70 of transitional cell carcinoma (TCC).
Results: DNA methylation on CpG islands of the p16 (0%, 17% and 21%) and death-associated protein kinase (13%, 33% and 29%) genes, and methylated in tumor-2 (56%, 60% and 76%), 12 (0%, 6% and 30%), 25 (25%, 27% and 35%) and 31 (45%, 56% and 79%) clones was detected in normal urothelium, NBCs and TCCs, respectively. The incidence of concurrent DNA hypermethylation on 3 or more CpG islands in NBCs (38%) was significantly higher than that in normal urothelium (0%, p = 0.0455) and even higher in TCCs (59%, p = 0.0043). The incidence of the CpG island methylator phenotype in nonpapillary carcinomas (nodular invasive carcinomas and their precursors, ie flat carcinoma in situ, 71%) was significantly higher than in papillary carcinomas (40%, p = 0.0143). In all specimens examined concurrent DNA hypermethylation on 3 or more CpG islands significantly correlated with immunohistochemically evaluated DNMT1 protein over expression (p = 0.0167).
Conclusions: DNA hypermethylation on multiple CpG islands in association with DNMT1 protein over expression may participate in multistage urothelial carcinogenesis even at the precancerous stage and particularly in the development of nodular invasive carcinomas of the bladder.

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5-Aza-2′-deoxycytidine restores the E-cadherin system in E-cadherin-silenced cancer cells and reduces cancer metastasis

Nam J., Ino Y., Kanai Y., Sakamoto M., Hirohashi S.

Clinical and Experimental Metastasis (Clinical and Experimental Metastasis) 21 ( 1 ) 49 - 56 2004.03

ISSN 02620898

Down-regulation of the E-cadherin-mediated cell adhesion system is strongly related to cancer invasion and metastasis. Aberrant CpG hypermethylation in the promoter region of the E-cadherin gene has been shown to be responsible for reduction of E-cadherin expression. The present study was designed to test the hypothesis that the demethylating agent 5-aza-2′-deoxycytidine (AZA) can restore the E-cadherin system and reduce the potential for metastasis. AZA treatment modified the methylation status of the 5′ CpG island in the E-cadherin promoter, and induced re-expression of E-cadherin in human cancer cells whose E-cadherin expression had been silenced. The re-expressed E-cadherin was correlated with increased in vitro aggregation and reduced motility. After inoculation of cancer cells (MDA-MB-435S) into the mammary fat pads of mice with severe combined immunodeficiency, the mice were treated for nine consecutive weeks with AZA three times per week i.p. The AZA treatment suppressed both growth of the primary tumor and lung metastasis in comparison with untreated controls, suggesting that the suppression of metastasis may be, at least partly, attributable to restoration of E-cadherin expression. Therefore, inhibition of DNA methylation may be useful for preventing cancer metastasis.

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Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis.

Kanai Y, Saito Y, Ushijima S, Hirohashi S.

J Cancer Res Clin Oncol (SPRINGER) 130 ( 11 ) 636 - 644 2004

Accepted, ISSN 01715216

Purpose: Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, correlates significantly with DNA hypomethylation in pericentromeric satellite regions, which is known to result in centromeric decondensation and enhanced chromosomal recombination in precancerous conditions and hepatocellular carcinomas (HCCs). We aimed to elucidate further the significance of DNMT3b4 during human hepatocarcinogenesis. Methods: DNMT3b4-transfected human epithelial 293 cells were characterized using growth rate measurements, gene expression microarray, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR was also performed on eight normal liver specimens, 45 noncancerous liver specimens showing chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 56 HCCs. Results: The growth rate of the DNMT3b4 transfectants was about double that of mock-transfectants. Induction of signal transducer and activator of transcription 1 (STAT1), an effector of interferon signaling, and of a set of downstream genes implicated in such signaling, was observed in the DNMT3b4 transfectants. There was significant correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs. mRNA expression levels of STAT1 and the three downstream genes examined were all significantly elevated in the chronic hepatitis and cirrhosis specimens compared with the normal liver specimens. Among the HCCs, the mRNA expression levels of STAT1 and the downstream genes were higher in tumors without portal vein involvement than in more malignant HCCs with portal vein involvement. Significant correlations between the mRNA expression levels of STAT1 and each of the downstream genes were observed in the tissue samples. Conclusions: Overexpression of DNMT3b4 is involved in human hepatocarcinogenesis, even at the precancerous stages, not only by inducing chromosomal instability but also by affecting the expression of specific genes.

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