Faculty Profiles - TOYOOKA Shinichi

Promoter methylation downregulates CDX2 expression in colorectal carcinomas Reviewed

Hiroshi Kawai, Kunitoshi Tomii, Shinichi Toyooka, Masaaki Yano, Masakazu Murakami, Kazunori Tsukuda, Nobuyoshi Shimizu

Oncology Reports 13 ( 3 ) 547 - 551 2005.3

Language：English Publishing type：Research paper (scientific journal)

CDX2 (caudal type homeobox transcription factor 2) is a homeobox protein, which is expressed in intestinal epithelium. CDX2 has been considered to play a role as a tumor suppressor gene in colorectal cancer because its expression is lacking in colorectal carcinomas, but preserved in adenomas. The point mutations have been found to account for loss of CDX2 expression, but the main mechanism responsible for CDX2 gene inactivation is less understood. We analyzed methylation and expression status of CDX2 in colorectal cancer cell lines and primary tumors. There are two CpG-rich sites in promoter region of CDX2 gene, -1570 to -1200 and -220 to +880. By COBRA (combined bisulfite restriction analysis) assay, the upper CpG-rich site was heavily methylated in all cell lines, but the lower CpG-rich site was methylated in limited cell lines. Bisulfite sequencing analysis and RT-PCR revealed that methylation of the lower CpG site was associated with down-regulation of CDX2. In addition, gene expression was restored in COLO201, a methylated cell line with 5-aza-2′-deoxycytidine, confirming that methylation caused gene down-regulation. We also examined CDX2 promoter methylation of primary tumors by MSP (methylated-allele specific PCR) assay and found that nearly 40% of cases have a methylated CDX2 gene. Our results demonstrate that CDX2 methylation is frequently present in colorectal cancers and may play a key role in inactivating CDX2 expression.

Somatic mutations of the HER2 kinase domain in lung adenocarcinomas Reviewed

H Shigematsu, T Takahashi, M Nomura, K Majmudar, M Suzuki, H Lee, Wistuba, I, KM Fong, S Toyooka, N Shimizu, T Fujisawa, JD Minna, AF Gazdar

CANCER RESEARCH 65 ( 5 ) 1642 - 1646 2005.3

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small. cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P = 0.02) and adenocarcinoma histology (2.8%, 11 of 394; P = 0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.

Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers Reviewed

H Shigematsu, L Lin, T Takahashi, M Nomura, M Suzuki, Wistuba, II, KM Fong, H Lee, S Toyooka, N Shimizu, T Fujisawa, ZD Feng, JA Roth, J Herz, JD Minna, AF Gazdar

JOURNAL OF THE NATIONAL CANCER INSTITUTE 97 ( 5 ) 339 - 346 2005.3

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS INC

Background. Mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers are associated with increased sensitivity of these cancers to drugs that inhibit EGFR kinase activity. However, the role of such mutations in the pathogenesis of lung cancers is unclear. Methods: We sequenced exons 18-21 of the EGFRTK domain from genomic DNA isolated from 617 non-small-cell lung cancers (NSCLCs) and 524 normal lung tissue samples from the same patients and 36 neuroendocrine lung tumors collected from patients in Japan, Taiwan, the United States, and Australia and from 243 other epithelial cancers. Mutation status was compared with clinicopathologic features and with the presence of mutations in KRAS, a gene in the EGFR signaling pathway that is also frequently mutated in lung cancers. All statistical tests were two sided. Results: We detected a total of 134 EGFR TK domain mutations in 130 (21%) of the 617 NSCLCs but not in any of the other carcinomas, nor in nonmalignant lung tissue from the same patients. In NSCLC patients, EGFR TK domain mutations were statistically significantly more frequent in never smokers than ever smokers (51% versus 10%), in adenocarcinomas versus cancer of other histologies (40% versus 3%), in patients of East Asian ethnicity versus other ethnicities (30% versus 8%), and in females versus males (42% versus 14%; all P<.001). EGFR TK domain mutation status was not associated with patient age at diagnosis, clinical stage, the presence of bronchioloalveolar histologic features, or overall survival. The EGFR TK domain mutations we detected were of three common types: in-frame deletions in exon 19, single missense mutations in exon 21, and in-frame duplications/insertions in exon 20. Rare missense mutations were also detected in exons 18, 20, and 21. KRAS gene mutations were present in 50 (8%) of the 617 NSCLCs but not in any tumors with an EGFR TK domain mutation. Conclusions: Mutations in either the EGFR TK domain or the KRAS gene can lead to lung cancer pathogenesis. EGFR TK domain mutations are the first molecular change known to occur specifically in never smokers.

DOI： 10.1093/jnci/dji055

Aberrant methylation profile of human malignant mesotheliomas and its relationship to SV40 infection Reviewed

Makoto Suzuki, Shinichi Toyooka, Narayan Shivapurkar, Hisayuki Shigematsu, Kuniharu Miyajima, Takao Takahashi, Victor Stastny, Andrea L. Zern, Takehiko Fujisawa, Harvey I. Pass, Michele Carbone, Adi F. Gazdar

Oncogene 24 ( 7 ) 1302 - 1308 2005.2

Language：English Publishing type：Research paper (scientific journal)

Malignant mesothelioma (MM) is associated with asbestos exposure and the presence of SV40 viral sequences. Recently, we reported that SV40 infection of human mesothelial cells (HM) causes aberrant methylation of the tumor suppressor gene (TSG) RASSF1A. We investigated methylation of 12 genes by methylation-specific PCR in 63 MMs, six MM cell lines, and two foci of SV40-infected HM. Methylation percentages of the tested genes ranged from 3 to 65%. The frequencies of HPP1, RASSF1A, Cyclin D2, and RRAD methylation, and the value of the methylation index, were significantly higher in SV40 sequence-positive MMs than in SV40-negative MMs. Methylation of TMS1 and HIC-1 was associated with shortened survival. SV40-infected HM showed progressive aberrant methylation of seven genes (RASSF1A, HPP1, DcR1, TMS1, CRBP1, HIC-1, and RRAD) during serial passage. Our results demonstrate a relationship between SV40 and methylation of multiple genes in MM, indicating that the virus plays a role in the pathogenesis of MM. © 2005 Nature Publishing Group. All rights reserved.

DOI： 10.1038/sj.onc.1208263

Aberrant methylation of HIN-1 (high in normal-1) is a frequent event in many human malignancies Reviewed

H Shigematsu, M Suzuki, T Takahashi, K Miyajima, S Toyooka, N Shivapurkar, GE Tomlinson, D Mastrangelo, HI Pass, E Brambilla, UG Sathyanarayana, B Czerniak, T Fujisawa, N Shimizu, AF Gazdar

INTERNATIONAL JOURNAL OF CANCER 113 ( 4 ) 600 - 604 2005.2

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers. We studied HIN-1 methylation status in many types of adult and pediatric malignancies and cell lines. We examined the expression of HIN-1 mRNA in 52 cell lines and the promoter methylation status in the cell lines and in over 800 primary tumors representing 17 tumor types using methylation specific PCR. Promoter methylation was observed in 73% of breast cancer, 67% of nonsmall cell lung cancer (NSCLC), 30% of small cell lung cancer (SCLC) and 57% of malignant mesothelioma (MM) cell lines, and methylation was completely correlated with loss of expression. Expression negative cell lines restored HIN-1 expression after treatment with 5-aza-2'-deoxycytidine. Promoter methylation of HIN-1 was found in 90% of retinoblastomas, 73% of Wilms' tumors, 61% of rhabdomyosarcomas, 57% of breast cancers, 52% of prostate cancers, 40% of MMs, 28% of NSCLCs and 27% of lymphomas. Methylation frequencies in colorectal cancers, cervical cancers, bronchial carcinoids, SCLCs, neuroblastomas, osteosarcomas, leukemia, medulloblastomas and bladder cancers were lower (4-21%), while hepatoblastomas lacked methylation. HIN-1 methylation was rarely detected in nonmalignant tissues (8 of 165, 5%). Aberrant methylation of HIN-1 with loss of expression is a common event and may contribute to the pathogenesis of many types of human malignancies.

DOI： 10.1002/ijc.20622

再発肺癌に対するラジオ波焼灼療法 (RFA) の有用性について(再発肺癌の治療, 第22回日本呼吸器外科学会総会)

佐野由文, 豊岡伸一, 青江基, 岡部和倫, 伊達洋至, 金澤右, 清水信義

日本呼吸器外科学会雑誌 19 ( 3 ) 316 - 316 2005

Atypical adenomatous hyperplasia 合併・非合併多発肺腺癌における臨床病理学的因子の検討(肺癌 (8), 第22回日本呼吸器外科学会総会)

豊岡伸一, 永井完治, 西條天基, 似鳥純一, 萩原優, 菱田智之, 塩野知志, 吉田純司, 西村光世, 伊達洋至, 清水信義, 石井源一郎, 西脇裕

日本呼吸器外科学会雑誌 19 ( 3 ) 427 - 427 2005

青江基, 豊岡伸一, 岡部和倫, 佐野由文, 伊達洋至, 清水信義

日本呼吸器外科学会雑誌 19 ( 3 ) 352 - 352 2005

Unilateral chronic rejection after living-donor lobar lung transplantation

The Journal of the Japanese Association for Chest Surgery 19 ( 3 ) 281 - 281 2005

W11-2 気管支鏡, 胸腔鏡を使った対策(<ワークショップ11>肺切除後合併症と対策)(第28回日本呼吸器内視鏡学会総会)

青江基, 豊岡伸一, 岡部和倫, 佐野由文, 伊達洋至, 清水信義

気管支学 27 ( 3 ) 197 - 197 2005

The relationship between aberrant methylation and survival in non-small-cell lung cancers Reviewed

S. Toyooka, M. Suzuki, R. Maruyama, K. O. Toyooka, K. Tsukuda, Y. Fukuyama, T. Iizasa, M. Aoe, H. Date, T. Fujisawa, N. Shimizu, A. F. Gazdar

British Journal of Cancer 91 ( 4 ) 771 - 774 2004.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

The present study examined the relationship between methylation of five genes (p16INK4a, RASSF1A, APC, RARβ and CDH13) and patient survival in 351 cases of surgically resected lung cancers. While there was no relationship between the other genes and survival, p16INK4a methylation was significantly related to unfavourable prognosis in lung adenocarcinomas. © 2004 Cancer Research UK.

DOI： 10.1038/sj.bjc.6602013

Less frequent promoter hypermethylation of DLC-1 gene in primary breast cancers Reviewed

A Teramoto, K Tsukuda, M Yano, S Toyooka, H Dote, H Doihara, N Shimizu

ONCOLOGY REPORTS 12 ( 1 ) 141 - 144 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Absence or low expression of DLC-1, a tumor suppressor gene, in breast cancers has been shown recently. LOH of 8p12-p22, on which DLC-1 is located, is frequent in breast cancers, but the correlation between low expression of DLC-1 and LOH has not been confirmed. To determine the implication of aberrant methylation, one of the most frequent mechanisms of silencing the tumor suppressor or cancer-related genes, we examined the methylation status of DLC-1 promoter region in breast cancer cell lines and primary breast tumors. The hypermethylation status was examined by MSP and 25% of cell lines harbored a methylated allele. The gene silencing by methylation was also confirmed by the reexpression of DLC-1 by the 5-aza-2'-deoxycytidine treatment in DLC-1 hypermethylated cell line. But the methylation of DLC-1 gene was less frequently shown in primary breast cancers (10%). These data suggest that hypermethylation is responsible for silencing of DLC-1 gene in a limited portion of breast cancers.

DNA methylation of multiple genes and clinicopathological relationship of non-small cell lung cancers Reviewed

T Hanabata, K Tsukuda, S Toyooka, M Yano, M Aoe, Nagahiro, I, Y Sano, H Date, N Shimizu

ONCOLOGY REPORTS 12 ( 1 ) 177 - 180 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Aberrant methylation of 5'CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in several types of cancers. In non-small cell lung cancer (NSCLC), several genes are known to be frequently methylated and the correlation of their methylation with clinical features has been studied. We determined the methylation of p16, CDH13 and RAR-beta which were reported to be methylated frequently in NSCLCs and HPP-1 which was known to be methylated in other types of cancers. The correlation between methylation and clinicopathological features were examined. The frequencies of methylation in NSCLCs were 20% for p16, 37% for CDH13, 34% for RAR-beta, and 13% for HPP1. The methylation of p16 is correlated with smoking history and methylation of HPP1 was significantly more frequent in adenocarcinomas than in squamous cell carcinomas. This is the first description of aberrant methylation of the HPP1 gene in lung cancers and our data support the previous reports on methylation in NSCLCs and association with smoking.

SYT-SSX fusion genes in synoviat sarcoma of the thorax Reviewed

M Yano, S Toyooka, K Tsukuda, H Dote, Y Morimoto, N Ohata, K Ichimura, M Aoe, H Date, N Shimizu

LUNG CANCER 44 ( 3 ) 391 - 397 2004.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

Synovial sarcoma (SS) is characterized by a chromosomal translocation resulting in the expression of an SYT-SSX chimeric transcript, usually SYT-SSX1 or SYT-SSX2. Synovial sarcoma typically originates in the limbs, and its location in the thorax is rare. Synovial sarcomas are usually classified into three histologic subtypes: biphasic, monophasic and poorly differentiated tumors. The detection of the characteristic chimeric transcript often contributes to a histopathological diagnosis, especially when the tumor arises in an unusual location. Previous studies have shown that SYT-SSX1 is the most common SYT-SSX fusion transcript in biphasic synovial sarcomas of the limbs. Here, we report two cases of synovial sarcoma originating in the thorax. The presence of SYT-SSX2 chimeric transcripts was confirmed by reverse transcript polymerase chain reaction (RT-PCR) and a direct sequencing analysis in both cases. The tumor in case 1 originated from the pericardium, which is an exceedingly rare site for primary synovial sarcoma; only three other cases of synovial sarcoma originating in the pericardium have been previously reported. Case 2 exhibited a biphasic synovial sarcoma of the mediastinum containing an SYT-SSX2 fusion transcript, which is a rare fusion type in biphasic synovial sarcomas of the limbs. We reviewed previous reports of thoracic synovial sarcomas containing an analysis of the SYT-SSX fusion transcript and found that case 2 in the present study was the first description of a biphasic synovial sarcoma of the thorax with an SYT-SSX2 fusion transcript. However, the number of reported cases was not sufficient to conclude that SYT-SSX2 fusion in biphasic synovial sarcoma of the thorax is, indeed, rare. Further genetic analysis is needed to fully understand the biological and clinical features of synovial sarcoma originating in the thorax. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.lungcan.2003.11.011

The relationship between NY-ESO-1 mRNA expression and clinicopathological features in non-small cell lung cancer Reviewed

J Konishi, S Toyooka, M Aoe, Y Omura, K Washio, K Tsukuda, N Shimizu

ONCOLOGY REPORTS 11 ( 5 ) 1063 - 1067 2004.5

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Tumor-associated antigens recognized by cellular or humoral effectors of the immune system are potential targets for antigen-specific cancer immunotherapy. NY-ESO-1 is one of the most immunogenic cancer/testis (CT) antigens and emerges as the potential candidate for specific immunotherapy. We studied mRNA expression status of NY-ESO-1 in 63 cases of NSCLCs using the real-time reverse transcription PCR to examine the relationship between its expression and clinicopathological features. NY-ESO-1 expression was present in 20 (32%) of 63 NSCLC cases and significantly increased with the advancement of disease stage in TNM classification (P=0.013), especially related to lymph node metastasis (P=0.020). Moreover, frequency of NY-ESO-1 expression was related to the degree of pathological differentiation (P=0.035). The quantity of NY-ESO-1 expression by real-time RT-PCR was not correlated with any clinicopathological factor. Our results demonstrate that the NY-ESO-1 expression was frequently present in primary NSCLC, especially advanced cases with lymph node metastasis. In addition, the high incidence of NY-ESO-1 expression in NSCLC suggests the possibility of a specific immunotherapy for NSCLC.

Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types Reviewed

N Shivapurkar, S Toyooka, KO Toyooka, J Reddy, K Miyajima, M Suzuki, H Shigematsu, T Takahashi, G Parikh, HI Pass, PM Chaudhary, AF Gazdar

INTERNATIONAL JOURNAL OF CANCER 109 ( 5 ) 786 - 792 2004.5

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 and DRS), and 2 potentially antiapoptotic receptors lacking death domains (DcRI and DcR2) Aberrant promoter methylation and resultant silencing of tumor suppressor genes play an important role in the pathogenesis of many tumor types. Recently aberrant methylation of TRAIL decoy receptors was reported in pediatric tumor cell lines and neuroblastomas. We examined the methylation and expression status of TRAIL receptor genes in cancers of breast, lung, mesothelioma, prostate, bladder, cervix, ovary, brain and in hematopoietic malignancies. Aberrant methylation of DcR1 or DcR2 was present in 70% of primary breast cancers, 31% of primary lung cancers, in 63% of primary malignant mesothelioma (MM), in 60% of prostate cancer, in 42% of bladder cancer, in 100% of cervical cancer, in 43% of ovarian cancer, in 41% of lymphoma, in 26% of leukemia and in 56% of multiple myeloma. Methylation of DR4 and DR5 was rare in all the tumor types examined. Methylation of all the 4 receptors was rare in non malignant tissues. In cell lines, aberrant methylation of DcR1 was present in 11 of 23 (48%) breast, 10 of 27 (37%) lung and 3 of 7 (43%) MM, whereas aberrant methylation of DcR2 was present in 17 of 23 (74%) breast, 13 of 27 (48%) lung and 5 of 7 (71%) MM. The concordance between loss of gene expression and aberrant methylation ranged from 70-100%. Treatment with 5-aza-2'-deoxycytidine restored DcR1 and DcR2 expression in 9 methylated cell lines confirming that aberrant methylation was the cause for silencing of DcRI and DcR2 expression. Our results demonstrate that DcRI and DcR2 genes are frequently methylated in various tumor types, and that the role of decoy receptors in tumor pathogenesis needs to be re-evaluated. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/ijc.20041

Mutation analysis of the BRAF codon 599 in malignant pleural mesothelioma by enriched PCR-RFLP Reviewed

H Dote, K Tsukuda, S Toyooka, M Yano, HI Pass, N Shimizu

ONCOLOGY REPORTS 11 ( 2 ) 361 - 363 2004.2

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

BRAF encodes a RAS-regulated serine/threonine kinase that mediates the pathway for cell growth and malignant transformation. Point mutations of BRAF were reported recently in 66% of melanomas, over 30% of thyroid papillary and low-grade ovarian cancers, and a smaller percentage of other human cancers. Mutations in malignant cells were reported to occur only in exons 11 and 15. Among these mutations, BRAF V599E is most frequent and proved to invert its transcript to the dominant active form. To exclude the interference of co-existing normal cells in clinical samples, we developed a new enriched PCR-RFLP assay for detecting mutations of BRAF codon 599 mutation. The sensitivity of this assay was examined to find that one mutant allele among C, 102 wild-type alleles could be detected. We applied this method for 53 cases of primary malignant pleural mesotheliomas (MPMs) and 6 cell lines and found no mutations in these samples. Our results demonstrate that the developed enriched PCR-RFLP is a sensitive assay to detect BRAF codon 599 mutation. However, it may be a rare type of mutation in MPMs. Our new assay is useful and can be applied for screening of BRAF codon 599 mutation in various kinds of clinical samples.

Primary Thymic Adenocarcinoma with Production of Carbohydrate Antigen 19-9 and Carcinoembryonic Antigen Reviewed

Takahiko Misao, Yosuke Yamamoto, Hideharu Nakano, Shinichi Toyooka, Masataka Yamane, Katashi Satoh

Japanese Journal of Thoracic and Cardiovascular Surgery 52 ( 1 ) 30 - 32 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：Japanese Association for Thoracic Surgery

A 59-year-old male, whose chest X-ray showed an abnormal shadow, visited us for further study. Laboratory examination showed the elevated level for both carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) in serum, and the chest X-ray and computed tomography showed an anterior mediastinal mass. Under a diagnosis of thymic malignancy, extended thymectomy with combined resection of the pleura and pericardium was performed. Histopathological findings showed a well-differentiated adenocarcinoma of the thymus, in which CA19-9 and CEA were positive immuno-histochemically. The level of serum CA19-9 and CEA returned to normal ranges postoperatively, however, the tumor recurred in local site with re-elevation of these tumor markers at the 20th month after surgery. He died at 4th month after the first recurrence despite the intensive chemotherapy. We report an extremely rare case of primary thymic adenocarcinoma with the production of CA19-9 and CEA.

DOI： 10.1007/s11748-004-0058-3