Aichi Cancer Center Research Institute

Scientific Report 2012 – 2013

Chikusa-ku, Nagoya 464-8681 Japan

(The Cover) The Aichi Cancer Center Research Institute Main Building Published by Dr. Taira Kinoshita President Aichi Cancer Center Research Institute 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan Telephone: 052-762-6111 Facsimile: 052-763-5233 Editorial Committee Dr. Masahiro Aoki (Division of Molecular Pathology) Dr. Keiichiro Sakuma (Division of Molecular Pathology) Dr. Hiroshi Kumimoto (Central Service Unit) Dr. Malcolm A. Moore, English Editor Ms. Hiromi Tamaki (Director's Office) Printed by Nagoya University COOP 1 Furoucho, Chikusa-ku, Nagoya 464-0814, Japan

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Contents Preface

Taira Kinoshita 1 Organization of the Aichi Cancer Center Research Institute 2 Scientific Reports Division of Epidemiology and Prevention

General Summary 5 1. Descriptive epidemiology 1.1. Differences in incidence and trends of hematologic malignancies in Japan

and the United States Chihara, D., Ito, H., Matsuda, M., Shibata, A., Katsumi, A., Nakamura,

S., Sobue, T.,Morton, LM., Weisenburger, DD., and Matsuo, K. 2. The hospital-based epidemiologic research program at Aichi Cancer Center

(HERPACC) study 2.1. Time to first cigarette and upper aerodigestive tract cancer risk in Japan

Matsuo, K., Gallus, S., Negri, E., Kawakita, D., Oze, I., Hosono, S., Ito, H., Hatooka, S., Hasegawa, Y., Shinoda, M., Tajima, K., La Vecchia, C., and Tanaka, H.

2.2. Time to first cigarette and lung cancer risk in Japan Ito, H., Gallus, S., Hosono, S., Oze, I., Fukumoto, K., Yatabe, Y., Hida, T.,

Mitsudomi, T., Negri, E., Yokoi, K., Tajima, K., La Vecchia, C., Tanaka H., and Matsuo K.

2.3. Polymorphisms in base excision repair genes are associated with endometrial cancer risk among postmenopausal Japanese women

Hosono, S., Matsuo, K., Ito, H., Hirose, K., Oze, I., Watanabe, M., Nakanishi, T., Tajima, K., and Tanaka, H.

2.4. Coffee and green tea consumption is associated with upper aerodigestive tract cancer in Japan

Oze, I., Matsuo, K., Kawakita, D., Hosono, S., Ito, H., Watanabe, M., Hatooka, S., Hasegawa, Y., Shinoda, M., Tajima, K., and Tanaka, H.

3. Tobacco control 3.1. Factors associated with increasing body weight after smoking cessation

therapy in Japan Tanaka, H., Taniguchi, C., Oze, I., Saka, H., Ito, H., Tachibana, K., Tokoro,

A., Nozaki, Y., Nakamichi, N., Suzuki, Y., and Suehisa, H. 3.2. Cigarette smoke inhalation and lung cancer risk: Case-control study in Japan Fukumoto, K., Ito, H., Park C., Tanaka, H., Matsuo, K., Tajima, K., and

Takezaki, T. Division of Oncological Pathology

General Summary 1. Peptide-based non-invasive molecular delivery system by developing novel

cancer cell-penetrating peptides (tumor-homing CPPs) for advanced medical

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applications Saito, K., Matsushita, M., and Kondo, E 2. Coxsackie and adenovirus receptor – a novel negative regulator of RhoA

associated kinase (ROCK) - is a critical regulator of survival and growth of oral squamous carcinoma cells

Saito, K., Sakaguchi, M., Iioka, H., Matsui, M., Nakanishi, H., Huh N., and Kondo, E.

3. Anti-tumor impact of p14ARF on gefitinib-resistant non-small cell lung cancers Saito, K., Takigawa, N., Ohtani, N., Iioka, H., Tomita, Y., Ueda, R., Fukuoka, J.,

Kuwahara, K., Ichihara, E., Kiura, K., and Kondo, E. 4. Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines Oshima, Y., Tanaka, H., Murakami, H., Ito, Y., Furuya, T., Kondo, E., Kodera,

Y., and Nakanishi H. 5. LGR5 is a marker of poor prognosis in glioblastoma and is required for survival

of brain cancer stem-like cells Nakata, S., Campos, B., Bageritz, J., Bermejo, JL., Becker, N., Engel, F., Acker,

T., Momma, S., Herold-Mende, C., Lichter, P., Radlwimmer, B., and Goidts, V. Division of Molecular Oncology

General Summary 1. LIM-domain protein AJUBA suppresses malignant mesothelioma cell

proliferation via the Hippo signaling cascade Tanaka, I., Osada, H., Fujii, M., Fukatsu, A., Hida, T., Horio, Y., Kondo, Y.,

Sato, A., Hasegawa, Y., Tsujimura, T., and Y Sekido 2. RASSF3 downregulation increases malignant phenotypes of non-small cell

lung cancer Fukatsu, A., Ishiguro, F., Tanaka, I., Kudo, T., Nakagawa, K., Shinjo, K.,

Kondo, Y., Fujii, M., Hasegawa, Y., Tomizawa, K., Mitsudomi, T., Osada, H., Hata, Y., Sekido, Y.

Division of Molecular Medicine

General Summary 1. Cell cycle deregulation contributes to acute transformation in chronic type

adult T-cell leukemia/lymphoma Yoshida, N., Karube, K., Utsunomiya, A., Tsukasaki, K., Imaizumi, Y.,

Taira, N., Uike, N., Umino, A., Arita, K., Katayama, M., Tsuzuki, S., Kinoshita, T., Ohshima, K., and Seto, M.

2. Generation of mouse models of lymphoid neoplasms using retroviral gene transduction of in vitro-induced germinal center B and T cells

Arita, K., Maeda-Kasugai, Y., Ohshima, K., Tsuzuki, S., Katayama, M., Karube, K., Yoshida, N., Sugiyama, T., and Seto, M.

3. Comprehensive gene expression profiles of NK cell neoplasms indicate vorinostat as an effective drug candidate

Karube, K., Tsuzuki, S., Yoshida, N., Arita, K., Kato, H., Katayama, M., Ko, YH., Ohshima, K., Nakamura, S., Kinoshita, T., and Seto, M.

4. Clonal heterogeneity of lymphoid malignancies is related to poor prognosis

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Katayama, M., Tagawa, H., Nakagawa, M., Fukuhara, N., Sivasundaram, S., Takeuchi, I. , Kasugai, Y., Arita, K., Yoshida, N., Kakiuchi, T., Karube, K., Tsuzuki. S, Nakamura, S., and Seto, M.

5. TEL (ETV6)-AML1 (RUNX1) initiates self-renewing fetal pro-B cells in association with a transcriptional program shared with embryonic stem cells in mice

Tsuzuki, S., and Seto, M. Division of Immunology

General Summary 1. Constitutively active autophagy causes a CTL epitope generation in

pancreatic cancer cells Demachi-Okamura, A., and Kuzushima, K. 2. Construction and molecular characterization of a T-cell receptor-like

antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H

Akatsuka, Y., Inaguma, Y., Akahori, Y., Murayama, Y., Shiraishi, K., Endoh, A., Demachi-Okamura, A., Hiramatsu, K., Nishimura, Y., Takahashi, To., Emi, N., and Kuzushima, K.

3. Pluripotent stem cell-derived proliferating myeloid cells as a possible basis for cellular vaccine cancer immunotherapy

Zhang, R., Liu, T., Maki, H., Kuzushima, K., and Uemura, Y. 4. Modification of IL-12p70/osteopontin balance in dendritic cells by ligand

activation of invariant NKT cells Zhang, R., Liu, T., Kuzushima, K., and Uemura, Y. Division of Virology

General Summary 1. Interaction between Basic Residues of Epstein-Barr Virus EBNA1 Protein

and Cellular Chromatin Mediates Viral Plasmid Maintenance Kanda, T. and Tsurumi, T.

2. Contribution of Myocyte Enhancer Factor 2 (MEF2) Family Transcription Factors to BZLF1 Expression in Epstein-Barr virus Reactivation from Latency Murata, T. and Tsurumi, T.

3. Heat shock protein 90 inhibitors repress latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma cells Murata, T. and Tsurumi, T.

4. Different Distributions of Epstein-Barr Virus Early and Late Gene Transcripts within Viral Replication Compartments Sugimoto, A., Sato, Y., Murata, T., Kanda, T., and Tsurumi, T.

5. Nuclear Transport of Epstein-Barr Virus DNA Polymerase is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90 Kawashima, D., Murata, T., Kanda, T., and Tsurumi T.

6. Epstein-Barr Virus Deubiquitinase Downregulates TRAF6-mediated NF-κB Signaling during Productive Replication Saito, S., Murata, T., Kanda, T., and Tsurumi, T.

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7. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication Narita, Y., Murata, T., Kanda, T., and Tsurumi, T.

8. HLA-restricted Presentation of WT1 Tumor Antigen in B-lymphoblastoid Cell Lines Established using a Maxi-EBV System Kanda, T., Kuzushima, K., and Tsurumi, T.

9. Epigenetic Histone Modification of the Epstein-Barr Virus BZLF1 Promoter during Latency and Reactivation in Raji Cells Murata, T., Kondo, Y., and Tsurumi, T.

Division of Molecular Pathology

General Summary 1. c-Myc and CDX2 mediate E-selectin ligand glycan expression in colon

cancer cells undergoing EMT Sakuma, K., Kannagi, R., and Aoki, M. 2. CDX Transcription Factors Positively Regulate Expression of PLEKHG1

in Intestinal Epithelium Aoki, M., Fujishita, T., and Taketo, MM. 3. Roles of the mTOR signaling in intestinal adenocarcinoma formation in

cis-Apc/Smad4 mutant mice Fujishita, T., Taketo, MM., and Aoki, M. Division of Biochemistry

General Summary 1. Emerging roles of ubiquitin-proteasome machinery in formation of

primary cilia Kasahara, K., Era, S., Kawamoto, E., Kawakami, Y., Kiyono, T., Kawamura, Y., Goshima, N., and Inagaki, M.

2. Cytokinetic failure induces aneuploidy and aging in vimentin phosphorylation deficient mice Tanaka, Hir., Matsuyama, M., Inoko, A., Goto, H., Yonemura, S., Kobori, K., Tanigawa, N., Hayashi, Y., Kondo, E., Itohara, S., Izawa, I. and Inagaki, M.

3. Perspective of Aurora A kinase as a therapeutic target for cancer Goto, H., Watanabe, N., Kobori, K., Inoko, A., Mochizuki, H., Togashi, T., Kisu, Y., Kawamura, Y., Kawakami, Y., Goshima, N., and Inagaki, M.

4. Novel cell cycle regulation through centrosomes Inoko, A., Inaba, H., He, D., Goto, H., Hayashi, Y., Izawa, I., Urano, T., Yonemura, S., Kiyono, T., Goshima, N., and Inagaki, M.

5. Interaction of Cell Polarity Regulator Scribble with Multidrug Resistance Protein 4 (MRP4/ABCC4) Izawa, I., Hayashi, Y., and Inagaki, M.

Division of Epigenomics

General Summary 1. The PRC2 chromatin regulator is a key regulator of epigenetic plasticity

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in glioblastoma Natsume, A., Ito, M., Katsushima, K., Ohka, F., Hatanaka, A., Shinjo, K., Sato, S., Takahashi, S., Ishikawa, Y., Takeuchi, I., Shimogawa, H., Uesugi, M., Okano, H., Kim, SU., Wakabayashi, T., Issa, JP., Sekido, Y., and Kondo, Y.

2. Hepatitis Virus Infection Affects DNA Methylation in Mice with Humanized Livers. Okamoto Y, Shinjo K, Shimizu Y, Sano T, Yamao K, Gao W, Fujii M, Osada H, Sekido Y, Murakami S, Tanaka Y, Joh T, Sato S, Takahashi S, Wakita T, Zhu J, Issa JP, and Kondo Y.

Central Service Unit

General Summary 1. Relationship between risk of esophageal cancer and the number of

polymorphisms in mitochondrial DNA Kumimoto, H.

Librarians T. Yasuda, T. Shibata, T. Matsunaga, M. Sakou, and N. Terashima Publications 1. Journals 2. Reviews and books 3. Abstracts for international conferences Records of seminars Author index for research reports and publications

From left to right Ms. H. Tamaki and Dr. T. Kinoshita Inset: Dr. K. Tajima

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Preface _______________________________________________________________________________________

It is my pleasure to share with you the 23rd Scientific Report (2012-2013) of the Aichi Cancer Center Research Institute. Since its establishment in 1964, Scientific Reports have been published biennially to document major research activities and highlight progress in and contributions to cancer research worldwide.

As illustrated on the following pages, the Research Institute consists of 8 Divisions, along with a Central Service Unit, an Animal Facility and a Laboratory of Translational Research, with overall research emphasis on three areas: cancer prevention/epidemiology; preclinical/experimental therapy; and carcinogenesis/molecular biology. In addition, the institute is affiliated with the Nagoya University Graduate School of Medicine and the Nagoya City University Graduate School of Pharmaceutical Sciences. At the present time 7 professors and 4 associated professors nominated from chiefs and section heads of the Research Institute are engaged in education of graduate school students at the two universities. In Aichi Cancer Center Research Institute itself, a total of 76 staff members, 42 researchers and 18 research assistants, as well as 16 research residents, are now conducting a wide range of studies, together with 8 graduate school students, and approximately 25 visiting research fellows and 40 temporary research assistants. The major areas being pursued are as follows: - descriptive and analytical epidemiology of cancers - primary and secondary prevention of cancer - molecular pathogenesis of intractable malignancies

- peptide-based anti-cancer DDS technology - molecular oncology of respiratory tract cancer - molecular biology of translocation-junction genes in hematopoietic tumors - basic studies for cancer immunotherapy - oncogenicity, molecular biology and immunology of DNA tumor viruses - glycobiology of cancer cells in relation to metastasis - genetic dissection of signaling pathways in colon cancer

- molecular mechanisms of cell proliferation and movement - involvement of repair mechanisms in carcinogenesis

More detailed descriptions of the individual research topics of each Division appear in the contents of this report. It is our sincere hope that the activities of the Institute will make a major contribution to elucidation of the mechanisms of carcinogenesis and to development of novel clinical applications in cancer diagnosis, treatment and prevention.

Finally, I would like to express my deep appreciation to the Aichi Prefectural Government for the continuous support received since this Institute was founded in 1964. Granting support from the Ministry of Education, Science, Sports, Culture and Technology, the Ministry of Health, Labor, and Welfare, the Ministry of Economy, Trade and Industry, Japan, and other related organizations, is also gratefully acknowledged.

January, 2014

Taira Kinoshita, M.D., M.P.H., D.M.Sci. Acting Director, President

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Organization of the Aichi Cancer Center Research Institute _______________________________________________________________________________________

SCIENTIFIC REPORTS

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From left to right

First row: Dr. I. Oze, Dr. H. Ito, Dr. K. Matsuo, Dr. H. Tanaka, Dr. S. Hosono, Ms. M. Watanabe Second row: Ms. Y. Yamauchi, Ms. K. Fukaya, Ms. A. Yoshida, Ms. S. Inui, Ms. A. Uematsu,

Ms. M. Miyoshi, Ms. Y. Matsubara, Ms. T. Ito Third row: Dr. K. Ishioka, Ms. S. Torii, Ms. Y. Tanaka, Ms. T. Nishiwaki, Ms. S. Irikura, Ms. R.

Saito, Ms. Y. Mano Fourth row: Dr. M. Nakao, Dr. K. Fukumoto, Dr. D. Chihara, Ms. I. Kato, Ms. S. Nimura, Ms.

M. Yamaguchi, Ms. A. Hiraiwa, Ms. Y. Hamajima, Ms. K. Sugamuma

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Division of Epidemiology and Prevention ________________________________________________________________________________ Hideo Tanaka, M.D., PhD. Chief Keitaro Matsuo, M.D., PhD., S.M. Section Head (until June 2013) Hidemi Ito, M.D., PhD., M.P.H. Section Head (as of April 2009) Satoyo Hosono, M.D., PhD. Senior Researcher (as of April 2009) Isao Oze, M.D. Senior Researcher (as of April 2012) Daisuke Kawakita, M.D. Research Resident (until March 2012) Makoto Nakao, M.D. Research Resident (until March 2013) Koichi Fukumoto, M.D. Research Resident (as of April 2012) Dai Chihara, M.D., PhD. Research Resident (as of April 2013) Miki Watanabe, MSc. Research Assistant (as of April 2006) Misako Nakahama. Research Assistant (until March 2012) Tomoko Ito. Research Assistant (as of April 2012) Yasuo Morishima, M.D. PhD. Researcher (as of April 2011) Keitaro Matsuo, M.D., PhD., S.M. Researcher (as of July 2013) Visiting Trainees Kiyonori Kuriki, B.P., D.M.Sc. University of Shizuoka Takakazu Kawase, M.D., PhD. Fred Hutchinson Cancer Research Center. Daisuke Kawakita, M.D., PhD. Nagoya City University Hideo Nakane, PhD. Toyota Central R&D Labs., Inc. Masao Nishira, M.D. Nagoya University Graduate School of Medicine. Tania Islam, M.D. Nagoya University Graduate School of Medicine. Dai Chihara, M.D. Nagoya University Graduate School of Medicine. Aiko Sueta, M.D. Kumamoto University Graduate School of Medicine. Makiko Higashi, M.D. Nagoya University Graduate School of Medicine. Koichi Fukumoto, M.D. Nagoya University Graduate School of Medicine. Kuka Ishioka, M.D. Nagoya University Graduate School of Medicine. Chie Taniguchi. Nagoya Medical Center. General Summary

The current research activities of the Division of Epidemiology and Prevention cover the following three major subjects: (1) descriptive epidemiology of cancer incidence, mortality and survival using data from the Aichi Prefectural Cancer Registry and other population-based registries in collaborative studies; (2) analytical epidemiology based on the hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC) to determine risk and protective factors for cancer, with a particular focus on gene-environmental interactions; and (3) epidemiologic studies for tobacco control.

Our main findings in 2012 and 2013 were that: 1) in a comparative study of incidence in hematologic malignancies between Japan and the United States from 1993-2008, the age-adjusted incidence in Japan was approximately one-half that in the US but has been increasing significantly, whereas no significant change was seen in the US; 2) nicotine dependence, as indicated by the time to first cigarette (TTFC) of the day, is associated with an increased risk of upper aerodigestive tract and lung cancers; 3) polymorphisms in base excision repair genes (XRCC1 and haplotype TG) are associated with endometrial cancer risk among postmenopausal Japanese women; 4) coffee intake might be linked with a decreased risk of upper aerodigestive tract cancer, whereas that of green tea might confer an increased risk; 5) younger age, non-varenicline use, presence of a comorbidity with nicotine dependence, high FTND scores and success of quitting smoking were significantly associated with weight gain among persons who received smoking cessation therapy in Japan; and 6) inhalation of cigarette smoke is an independent risk factor for lung cancer after adjustment for pack-years of smoking in the Japanese population.

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1. Descriptive epidemiology 1.1 Differences in incidence and trends of

hematologic malignancies in Japan and the United States

Chihara, D., Ito, H., Matsuda, M.*1, Shibata, A.*1, Katsumi, A.*2, Nakamura, S.*3, Sobue, T.*4, Morton, LM.*5, Weisenburger, DD.*6, Matsuo, K.*7

The incidence of a malignant disease reflects genetics and cumulative exposure to factors in the living environment of any particular population. Therefore, evaluation of incidences and trends of disease in different populations may provide insights into its etiology and pathogenesis. With a focus on hematologic malignancies according to specific subtypes, we used population-based registry data in Japan (N=125,148) and the United States (US; N=172,925) from 1993 to 2008. Overall age-standardized incidence rates for all hematologic malignancies per 100,000 in 2008 were 18.0 for males and 12.2 for females in Japan, and 34.9 for males and 23.6 for females in the US. The age-adjusted incidence of hematologic malignancies in Japan was approximately one-half that in the US but has been increasing significantly, whereas no significant change was seen in the US {annual percent change (95%CIs): Japan, +2.4% (1.7, 3.1); US, +0.1% (-0.1, 0.2)}. The most frequent hematologic malignancy in both countries is NHL, accounting for 39.6% of all hematologic malignancies in Japan and 54.5% in the US. The lowest incidence was seen for CML, with respective figures of 3.4% and 3.0%. The incidences of lymphoid malignancies such as HL, NHL and MM showed marked differences between the US and Japan, whereas those for AML, ALL, and CML were less pronounced (Figure 1). HL and NHL are increasing substantially in Japan but not in the US, suggesting that environmental influences, such as Westernization of the life style, may be causing this increase. Differences in the incidence and trends for specific subtypes also showed a marked contrast across subtypes which, in turn, may provide significant new insights into disease etiology in the future. *1 Surveillance Division, Center for Cancer Control and Information Services, National Cancer Center, Japan. *2 Department of Clinical Oncology, Hamamatsu University School of Medicine, Hamamatsu, Japan *3 Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan. *4 Department of Environmental Medicine and Population Science, Osaka University Graduate School of Medicine, Osaka, Japan *5 Division of Cancer Epidemiology and Genetics,

National Cancer Institute, NIH, DHHS, Rockville, MD, USA *6 Department of Pathology, City of Hope National Medical Center, Duarte, CA, USA *7 Department of Preventive Medicine, Kyushu University Faculty of Medical Science, Fukuoka, Japan

2. The hospital-based epidemiologic re-

search program at Aichi Cancer Center (HERPACC) study

2.1 Time to first cigarette and upper

aerodigestive tract cancer risk in Japan Matsuo, K., Gallus, S.*1, Negri, E.*1, Kawakita, D., Oze, I., Hosono, S., Ito, H., Hatooka, S.*2, Hasegawa, Y.*3, Shinoda, M.*4, Tajima, K.*5, La Vecchia, C.*1, 6, Tanaka, H.

Cigarette smoking is the major cause for upper aerodigestive tract (UADT) cancers. The time to first cigarette (TTFC) of the day is a distinct indicator of nicotine dependence, but scanty information is available on its possible relation with UADT cancers (oral, oropharyngeal, hypopharyngeal, laryngeal, nasopharyngeal, and esophageal).

This case-control study included a total of 1,009 incident UADT cancer cases and 3,027 age- and sex-matched noncancer controls admitted to the Aichi Cancer Center (Nagoya, Japan) between 2001 and 2005. We estimated OR and 95% confidence intervals (CI) for TTFC using logistic regression models after adjustment for several potential confounders.

TTFC was inversely related to the risk of UADT cancer, and this association was consistent across subtypes of head and neck cancer and

Fig. 1. Incidences and trends for haematological malignancies from 1993 to 2008 in Japan (in blue) and the US (in red). Circles indicate the observed age-standardized incidence rates of males and females combined, and lines indicate the age-standardized incidence rates estimated by Joinpoint regression analysis. The axis denotes the annual incidence /100,000.

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esophageal cancer. For all UADT cancers considered among ever smokers and after accurate allowance for smoking quantity and duration, besides other relevant covariates, compared with TTFC more than 60 minutes, the adjusted ORs were 1.40 (95% CI: 0.93-2.11) for 31 to 60 minutes, 1.76 (95% CI: 1.20-2.58) for 6 to 30 minutes, and 2.43 (95% CI: 1.64-3.61) for within 5 minutes. No significant heterogeneity was found in strata of sex, age, alcohol consumption, fruit and vegetable intake, and occupation for overall and site-specific analysis.

Nicotine dependence, as indicated by the TTFC, is associated with increased risk of UADT cancers and is therefore an independent marker of exposure to smoking. Our result points to more detailed risk evaluation of UADT cancers allowed by the TTFC. *1 Department of Epidemiology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy *2 Respiratory Surgery, Aichi Cancer Center Central Hospital *3 Head/Neck Surgery, Aichi Cancer Center Central Hospital *4 Aichi Cancer Center Central Hospital *5 Aichi Cancer Center Research Institute *6 Department of Clinical Medicine and Community Health, Universitá degli Studi di Milan, Milan, Italy 2.2. Time to first cigarette and lung cancer

risk in Japan Ito, H., Gallus, S.*1, Hosono, S., Oze, I., Fukumoto, K., Yatabe, Y.*2, Hida, T.*3, Mitsudomi, T.*4, Negri, E.*1, Yokoi, K.*5, Tajima, K.*6, La Vecchia, C.*1,7, Tanaka H., Matsuo K.

Cigarette smoking is the major cause of lung cancer (LC). Although the time to first cigarette (TTFC) of the day is a distinct indicator of nicotine dependence, little information is available on its possible relation to LC.

This case-control study included a total of 1,572 incident LC cases and 1,572 non-cancer controls visiting for the first time the Aichi Cancer Center Hospital between 2001 and 2005. We estimated the odds ratio (OR) and 95% confidence intervals (CI) for TTFC using a logistic regression model after adjustment for several potential confounders.

TTFC was inversely associated with the risk of LC. This association was consistent across histological subtypes of LC. For all LCs considered among ever smokers and after accurate allowance for smoking quantity and duration, besides other relevant covariates, compared with TTFC >60 min, the adjusted ORs were 1.08 (95% CI, 0.73-1.61) for TTFC of 31-60 min, 1.40 (0.98-2.01) for 6-30 min and 1.86 (1.28-2.71) for within 5 min (Ptrend, < 0.001). Statistically marginally significant heterogeneity by histological subtype was observed (Pheterogeneity, 0.002).

Nicotine dependence, as indicated by the TTFC, is associated with increased risk of LC and is therefore can be considered an independent marker of exposure to tobacco smoking. *1 Department of Epidemiology, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan, Italy *2 Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Central Hospital *3 Department of Thoracic Oncology, Aichi Cancer Center Central Hospital *4 Department of Surgery, Division of Thoracic Surgery, Kinki University Faculty of Medicine, Osaka-Sayama *5 Department of Thoracic Surgery, Nagoya University Graduate School of Medicine, Nagoya *6 Aichi Cancer Center Research Institute *7 Department of Clinical Medicine and Community Health, Universitá degli Studi di Milan, Milan, Italy

Table 1. Associations between TTFC and UADT cancers among ever- and current smokers stratified by subsite. (Footnote: ORs were calculated by unconditional logistic regression adjusted for smoking status, duration of smoking, cigarettes per day, alcohol consumption, fruit and vegetable intake, and SES.)

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2.3. Polymorphisms in base excision repair genes are associated with endometrial cancer risk among postmenopausal Japanese women

Hosono, S., Matsuo, K.*1, Ito, H., Hirose, K.*2, Oze, I., Watanabe, M., Nakanishi, T.*3, Tajima, K.*4, Tanaka, H.*1

Objectives: Polymorphisms in base excision repair (BER) genes are associated with risk for several types of cancers but have not been studied with respect to endometrial cancer among Japanese. Therefore, we conducted a case-control study to explore the association between polymorphisms in base excision repair (BER) genes and the risk of endometrial cancer.

Methods/materials: This study included a total of 91 postmenopausal subjects with endometrial cancer and 261 non-cancer controls who visited Aichi Cancer Center between 2001 and 2005. We focused on single nucleotide polymorphisms (SNPs) within coding regions of five BER genes (OGG1, MUTYH, XRCC1, APEX1, PARP1). To assess lifestyle factors in the etiology of endometrial cancer, we used a self-administered questionnaire. Associations were evaluated using multivariate unconditional logistic regression models. We also assessed whether there were intergenic associations or any interaction with obesity.

Results: We observed a significant association between endometrial cancer risk and XRCC1 rs1799782 (C>T, Arg194Trp) and XRCC1 rs25487 (G>A, Arg399Gln), also uncovering a significant association between obesity (Body mass index

(BMI) ≥ 25) and rs25487. The XRCC1 polymorphisms were in complete linkage disequilibrium, and the XRCC1 TG haplotype was significantly associated with endometrial cancer risk. The interaction between the CA haplotype and BMI was marginally significant, whereas that between haplotype in XRCC1 and rs1136410 (PARP1) was not significant.

Conclusion: We found a significant association between endometrial cancer risk and XRCC1 polymorphisms and the TG haplotype in postmenopausal Japanese women. *1 Department of Epidemiology, Nagoya University Graduate School of Medicine *2 Department of Planning and Information, Aichi Prefectural Institute of Public Health *3 Department of Gynecologic Oncology, Aichi Cancer Center Hospital *4 Aichi Cancer Center Research Institute 2.4. Coffee and green tea consumption is

associated with upper aerodigestive tract cancer in Japan

Oze, I., Matsuo, K.*1, Kawakita, D.*2, Hosono, S., Ito, H., Watanabe, M., Hatooka, S.*3, Hasegawa, Y.*4, Shinoda, M.*5, Tajima, K.*6, Tanaka, H.

The impact of coffee and green tea consumption on upper aerodigestive tract (UADT) cancer risk has not been established in detail. Evaluation of the possible anti-carcinogenic properties of their ingredients is confounded by the potential increase in risk due to the high temperatures at which these beverages are generally consumed. We conducted a case-control study to evaluate the association

Table 2. Associations between FTTC and lung cancers (LCs) among ever and current smokers stratified by histologic subtypes. (Footnote: ORs were calculated by an unconditional logistic regression model adjusted for age, alcohol consumption, fruit and vegetable intake and socioeconomic status. Heterogeneity testing was carried out by likelihood-ratio test after estimations by an unconditional logistic regression model adjusted for age, sex, CPD, duration of smoking, smoking status, alcohol consumption, fruit and vegetable intake and occupation, except for a stratifying factor).

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between coffee and tea consumption and the risk of UADT cancer. The study enrolled 961 patients with UADT cancer and 2,883 non-cancer outpatients who visited Aichi Cancer Center between 2001 and 2005. Information on coffee and green tea consumption and other lifestyle factors was collected via a self-administered questionnaire. Consumption of 3 or more cups of coffee per day had a significant inverse association with UADT cancer (OR 0.73, 95% CI 0.55-0.96). In contrast, consumption of three or more cups of green tea per day had a significant positive association (OR 1.39, 95% CI 1.13-1.70). These links were evident for head and neck cancer but not for esophageal cancer. The association of coffee consumption with head and neck cancer was observed only among never smokers and alcohol drinkers. Similarly, the association of green tea consumption was observed among never smokers and never alcohol drinkers. No change was seen on stratification for confounding factors. These findings suggest that consumption of coffee might be associated with a decreased risk of UADT cancer, whereas that of green tea might confer increased risk. *1 Department of Preventive Medicine, Kyushu University Graduate School of Medicine *2 Department of Otorhinolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences *3 Department of Respiratory Surgery, Ichinomiya Nishi Hospital *4 Department of Head/Neck Surgery, Aichi Cancer Center Hospital *5 Department of Thoracic Surgery, Aichi Cancer Center Hospital *6 Department of Public Health and Occupational Medicine, Mie University Graduate School of Medicine 3. Tobacco control 3.1. Factors associated with increasing

body weight after smoking cessation therapy in Japan

Tanaka, H., Taniguchi, C.*1, Oze, I., Saka, H.*1, Ito, H., Tachibana, K.*2, Tokoro, A.*2, Nozaki, Y.*3, Nakamichi, N.*3, Suzuki, Y.*4, Suehisa, H.*5

Post-cessation weight gain possibly attenuates motivation to engage or sustain a smoking quit attempt that induces poorer cessation outcomes. Weight control intervention in patients who attempt to quit the habit is therefore an important issue. We aimed to identify factors associated with weight gain in smokers who received smoking cessation therapy (SCT) in Japan.

We observed weight change in 283 smokers

between the first visit of SCT and 12 months after the end of SCT. They were recruited from six hospitals between 2008 and 2011. We collected demographic information and presence of a comorbidity with nicotine dependence, as well as smoking history and the Brinkman Index obtained using self-report questionnaires at the first session. The subjects were followed up at 3, 6 and 12 months after the end of SCT, to obtain data on body weight and smoking status using a mail-based self-reported questionnaire. Risk of weight gain of +3.5 kg was assessed by stepwise logistic regression analysis.

Age 50 yr and over (OR, 0.38, 95%CI: 0.19-0.76) and varenicline use (OR, 0.30, 95%CI: 0.11-0.78) were significantly associated with low risk of weight gain. The presence of a comorbidity with nicotine dependence (OR, 3.33, 95%CI: 1.10-10.00), high level of nicotine dependence at baseline (OR, 2.07, 95%CI: 1.09-3.92) and success of quit smoking at 12 months after the end of SCT (OR, 4.57, 95%CI: 1.94-10.08) were significantly associated with weight gain.

Our study showed that younger age, non-varenicline use, presence of a comorbidity with nicotine dependence, high FTND scores and success in quitting smoking were significantly associated with weight gain among persons who received SCT in Japan. *1 National Hospital Organization Nagoya Medical Center *2 National Hospital Organization Kinki-Chuo Chest Medical Center *3 Social Insurance Chukyo Hospital *4 Kitasato University Kitasato Institute Hospital *5 National Hospital Organization Shikoku Cancer Center 3.2. Cigarette smoke inhalation and lung

cancer risk: Case-control study in Japan

Fukumoto, K., Ito, H., Park C., Tanaka, H., Matsuo, K.*1, Tajima, K.*2, Takezaki, T.*3

Several epidemiologic studies have demonstrated that degree of cigarette smoke inhalation is associated with the risk of lung cancer (LC) in European population. However, to date, there have been few such studies in Asian populations. Our aim was to clarify the relationship between cigarette smoke inhalation and the risk of LC in Japanese.

We therefore conducted a case-control study of cigarette smoking and LC risk in Aichi Prefecture. Between 1993 and 1998, several hospitals recruited newly diagnosed incident patients with histologically confirmed LC (n=653). The case

10

eligibility included an age of 20–81 years, no previous diagnosis of lung, or other cancer, and an ability to participate and provide informed consent. The study included both hospital controls (HC: n=453) and community-based controls (CC: n=828). The eligibility criteria for the two control groups were the same as for cases except that the hospital controls were admitted for non-malignant diseases or conditions unrelated to cigarette smoking. Odds ratios (OR) and 95% confidence intervals (CI) were derived from unconditional logistic regression analysis, adjusted for basic confounding variables such as age, sex, drinking status, family history of LC, occupation, and years of education.

The mean of age was 60.7 in cases and 60.1 in controls. Cases had a higher proportion of males, individuals having a family history of LC, heavy drinkers, and current smokers. The most common histologic types of LC were adenocarcinoma (AD) (50.9%), squamous cell carcinoma (SQ) (22.1%) and small cell carcinoma (SM) (19.2%). Compared to ever smokers in controls, those in cases started smoking at younger age, smoked more cigarettes per day and had smoked for a longer duration. The proportion of smokers who inhaled cigarette smoke was greater in cases.

The risk of LC in ever smokers was significantly higher than never smokers, especially in smokers who inhaled. Compared with never smokers, ORs for ever smokers who do not inhale cigarette smoke (inhale-no) and ever smokers who inhale cigarette smoke (inhale-yes) were 1.72 (95%CI: 1.15-2.59) and 3.28 (95%CI: 2.38-4.53), when adjusted for basic confounding variables. Stratified analyses by histologic types of LC showed that these associations were quite strong in SQ and SM (inhale-no: OR 10.1(95%CI: 4.96-20.7), inhale-yes: OR 19.7 (95%CI: 10.3-37.6)) On the other hand, such associations seemed to be mild in AD (inhale-no: OR 0.87(95%CI: 0.51-1.48), inhale-yes: OR 1.50(95%CI: 1.01-2.21)). Stratified analysis by pack-year showed that the OR of the “inhale-yes” group was higher than for the “inhale-no” group in every category of pack-years (<20, 20-39, 40-59, 60 or more).

When the analysis was restricted to ever smokers, the OR (adjusted for basic confounding factors and pack-year) of LC risk in ever smokers who inhaled cigarette smoke was significantly higher than who did not. OR for “inhale-yes” group compared with “inhale-no” group was 1.52 (95%CI: 1.06-2.18, p=0.021). No heterogeneity was observed across histologic types of LC. Our case-control study demonstrated that inhalation of cigarette smoke is an independent risk for LC

even after adjustment for pack-years in the Japanese population. Cigarette smoke inhalation might have an additive effect toward LC risk. *1 Department of Preventive Medicine, Kyushu University Faculty of Medical Science *2 Department of Public Health & Occupational Medicine, Mie University Graduate School of Medicine *3 Department of Island and Community Medicine, Kagoshima University Graduate School of Medical and Dental Sciences

Fig. 2. Association between cigarette

smoke inhalation and lung cancer risk in ever smokers

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From left to right

First row: Dr. Ken Saito, Dr. Akiko Yusa, Dr. Eisaku Kondo, Dr. Hayao Nakanishi, Dr. Susumu Nakata, Second row: Ms. Noriko Saito, Ms. Mayumi Yoshimura, Ms. Yumi Matsumoto, Ms. Risayo Watanabe Third row: Dr. Makoto Toneri, Dr. Hidekazu Iioka, Dr. Daisuke Yamashita, Dr. Takuya Saito,

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Division of Oncological Pathology ________________________________________________________________________________ Eisaku Kondo, M.D., Chief Hayao Nakanishi, M.D., Section Head Susumu Nakata, M.D., Senior Researcher Keiko Shinjo, M.D., Senior Researcher Ken Saito, Ph.D., Researcher Daisuke Yamashita, M.D., Research Resident (as of April 2013) Noriko Saito, Research Assistant Mayumi Yoshimura, Semi-regular Employee Keiko Nishida, Semi-regular Employee Risayo Watanabe, Semi-regular Employee (as of February 2013) Yuko Saito, Semi-regular Employee (until December 2012) Junya Yamamoto, Semi-regular Employee (until December 2012) Visiting Trainees Hidekazu Iioka, Ph.D., Laboratory of Bioregulatory Science, Advanced Medical Research Center, Aichi Medical University (as of May 2012) Akiko Yusa, Ph.D., Knowledge Hub Aichi, Priority Research Projects, Aichi Science & Technology Foundation Shinichiro Maseki, M.D., Dept. of Otorhinolaryngology, Konan Kosei Hospital Takuya Saito, M.D., Dept. of Surgery, Pref. Osaka Saiseikai Izuo Hospital Akihiro Ito, Dep. of Pharmacology, Konan Kosei Hospital Tomomi Furuya, Graduate School of Pharmaceutical Sciences, Meijo University (until September 2013) Kayoko Terazawa, Graduate School of Pharmaceutical Sciences, Meijo University (as of February 2013) Yumi Matsumoto, Dept. Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University (as of May 2013) Makoto Toneri, M.D., Dept. Anesthesia, Aichi Cancer Center Hospital (as of October 2012) General Summary

The aim for our research at the Division of Oncological Pathology is to disclose the pathogenesis of human malignancies, especially focusing on intractable tumors such as advanced staged cancers of gastrointestinal tract, oral, head and neck cancers, lung cancers, brain tumors, aggressive leukemias and so on, through molecular analyses based on the data obtained by morphological and biological examination of cancer tissues and human cancer cells. Our interest is now concretely concentrating on finding novel therapeutic targets through basic pathological research for molecular analysis of progression, metastasis, invasion, and stem cell pathology. As a second important aim, we are also making effort to develop advanced antitumor medical technologies for clinical application. In addition to these molecular pathological research activities, another important responsibility of our division is an autopsy service, which provides a basis with implications and providing motivation for new research activity. Postmortem examinations give us valuable information on the behavior of neoplasms and their response to therapy, helping clarify pathogenesis of cancers. Thus, the present aim of our division is promoting comprehensive pathological research and contributing to current and near-future tumor medicine. 1. Peptide-based non-invasive molecular delivery system by developing novel cancer cell-penetrating peptides (tumor-homing CPPs) for advanced medical applications Saito, K., Matsushita, M.*1, and Kondo, E.

Molecular targeting medicine has been recently supported by introduction of various anticancer

drugs such as small molecule inhibitors, chimeric antibodies and siRNAs. However, in tumor medicine, efficient drug delivery systems that enable specific targeting of tumors and minimize side effects have yet to be fully developed. We are now focusing on establishing peptide-based DDS with novel cell-penetrating peptides (CPPs), which

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are highly absorbable by human tumor cells, such peptides having non-invasive properties in vivo. Recently we generated tumor-lineage-homing CPPs that are highly permeable to cancer cells according to their tumor origins by employing a specific random peptide library. These CPPs are encoded by a novel artificial amino acid sequence and differ from conventional CPPs such as TAT, pAnt (antennapedia), and HIV gp41. For example, we obtained a CPP readily penetrating cells derived from colon adenocarcinomas, as well as examples for human leukemia and hepatocellular carcinoma cells. These tumor-lineage-homing CPPs should find application in peptide-based non-invasive medical technologies such as anti-cancer therapeutics and diagnostic imaging. *1 Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus 2. Coxsackie and adenovirus receptor – a

novel negative regulator of RhoA associated kinase (ROCK) - is a critical regulator of survival and growth of oral squamous carcinoma cells

Saito, K., Sakaguchi, M.*1, Iioka, H., Matsui, M., Nakanishi, H., Huh N.*1, and Kondo, E.

Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells. Constitutive expression in various cancerous and normal tissues has been reported and recently the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological functions in tumor cells remain controversial. We established a critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via specific interaction with Rho-associated protein kinase (ROCK). In particular, loss of endogenous CAR expression by knockdown using specific siRNA facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The

consequent morphological reaction appears reminiscent of anoikis, rather than epithelial-mesenchymal transition (EMT), and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix (ECM), but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membranes to cytoplasm. Immunoprecipitation assays of the

CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632), revealed that CAR directly associates with ROCKI and ROCKII, resulting in inhibition of ROCK activity and contributing to maintenence of cell-to-cell adhesion for growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. We found that the mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with control siRNA-pretreated cells that maintained endogenous CAR expression and that disseminated extensive peritoneal lesions. Thus, CAR substantially impacts on growth and survival of oral SCC cells through specific interactions with ROCK in vitro and in vivo, providing clues for the molecular therapy or head and neck SCCs.

*1 Department of Cell Biology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University

Fig. 1. Unique biological characteristics of CPPs as peptides (left). In vivo tumor detection by administration of novel tumor-homing CPPs (right). This report was published in Nature Commun., 3:951-963, July17, 2012.

Fig. 2. Schematic representation of biological effect of CAR (Coksackie and Adenovirus Receptor) on Rho A-associated kinase. Moderate suppression of ROCK activity by CAR facilitates growth, invasion and metastasis of SCC cells. This work was published in “Oncogene” March 18, 2013 [Epub ahead of print].

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3. Anti-tumor impact of p14ARF on gefitinib-resistant non-small cell lung cancers

Saito, K., Takigawa, N.,*1 Ohtani, N.,*2 Iioka, H., Tomita, Y., Ueda, R.,*3 Fukuoka, J., Kuwahara, K., Ichihara, E.,*4 Kiura, K.*4, and Kondo, E.

Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates cell proliferation. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as molecular-targeting agents for inactivation of EGFR signaling and have shown considerable therapeutic effects in non-small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. In this study, we aimed to characterize distinct molecular responses to gefitinib between drug-resistant and drug-sensitive lung adenocarcinoma cells. From quantitative PCR analysis, we found a specific increase in p14ARF expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14ARF triggered the most apoptosis in both clones. We further identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14ARF (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of gefitinib-resistant clones without affecting non-neoplastic cells. Notably, transduction of a minimized dose of the “p14 38-65 peptide” restored the response to gefitinib to that in the sensitive clones. These findings suggest that the region of p14ARF 38-65 a.a. is critical in the pharmacological action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers. *1 Department of General Internal Medicine 4, Kawasaki Medical School *2 Division of Cancer Biology, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR) *3 Department of Tumor Immunology, Aichi Medical University School of Medicine *4 Department of Respiratory Medicine, Okayama University Hospital

4. Lapatinib sensitivities of two novel

trastuzumab-resistant HER2 gene- amplified gastric cancer cell lines

Oshima, Y.*1, Tanaka, H., Murakami, H.*1, Ito, Y.*2, Furuya, T., Kondo, E., Kodera, Y.*1, and Nakanishi H.

Trastuzumab (Tmab) resistance is a major clinical problem needing resolution in patients with HER2-positive gastric cancers. However, in contrast to the situation for HER2-positive breast cancer lines, Tmab-resistant gastric cancer preclinical models that are needed to develop novel therapies to overcome this problem have not been available. We developed three new cell lines from HER2 gene-amplified gastric cancer cell lines (GLM-1, GLM-4, NCI N-87) by a new in vivo selection method consisting of repeated culture of small residual peritoneal metastases but not subcutaneous tumors after Tmab treatment. We then evaluated the anti-tumor efficacy of lapatinib in these Tmab-resistant cells. We successfully isolated two Tmab-resistant cell lines (GLM1-HerR2, GLM4-HerR2) among the three tested cell lines. The resistant cells differed from their parents in their flat morphology and rapid growth in vitro, whereas HER2, P95HER2 expression and Tmab binding were essentially the same in both parental and resistant cells. MUC4 expression was up- or down-regulated depending on the cell line. The resistant cells were still sensitive to lapatinib, similar to the parental cells, in vitro. The growth inhibition of the Tmab-resistant cells by lapatinib was due to both G1 cell-cycle

Figure 3. Specific gene regulation downstream of activated EGFR in lung adenocarcinomas in response to gefitinib. p14ARF, a tumor suppressor gene, is recruited on treatment with gefitinib to EGFR-activated lung cancer cells, with induction abrogated in gefitinib-resistant lung cancers derived from the sensitive parent cancer cells. The p1438-65 peptide has potential as an antitumor tool for gefitinib-resistant lung cancers. This report was highlighted in Molecular Cancer Therapeutics, an AACR journal. (Mol. Cancer Ther. 12:1616-1628, 2013)

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arrest and apoptosis induction via effective blockade of the PI3K/Akt and MAPK pathways. A preclinical study confirmed that the Tmab-resistant tumors are significantly susceptible to lapatinib. These results suggest that lapatinib has antitumor activity against Tmab-resistant gastric cancer cell lines, these latter being useful for understanding the mechanism of Tmab resistance and for developing new molecular therapeutic approaches. *1

Dep. of Gastroenterological Surgery, Nagoya University Graduate School of Medicine *2

Dep. of Gastroenterological Surgery, Aichi Cancer Center Central Hospital 5. LGR5 is a marker of poor prognosis in

glioblastoma and is required for survival of brain cancer stem-like cells

Nakata, S., Campos, B.*1, Bageritz, J.*2, Bermejo, JL.*3, Becker, N.*2, Engel, F.*2, Acker, T.*4, Momma, S.*5, Herold-Mende, C.*1, Lichter, P.*2, Radlwimmer, B.*2, and Goidts, V.*2

With various types of cancers including glioblastoma, there is accumulating evidence of the existence of cancer stem-like cells (CSCs), characterized by stem cell marker expression, capacity for differentiation and self-renewal, and a high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in various adult tissues, including intestinal mucosa and hair follicles in the skin. As Wnt signaling is also involved in brain development, we investigated the function of LGR5 in the maintenance of brain CSCs. Our study showed that the LGR5 transcript level was increased in brain CSCs in vitro, and in CD133-positive glioblastoma cells in tumor tissues. Functionally, silencing of LGR5 by lentiviral shRNA-mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression, which has been shown to be indispensible for brain CSCs. In line with an important function in glioma tumorigenesis, LGR5 expression could be shown to be increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs. *1

Div. of Neurosurgical Research, Dep. of Neurosurgery, University of Heidelberg *2

Div. of Molecular Genetics, German Cancer Research Center, Heidelberg *3

Institute of Medical Biometry and Informatics, University Hospital Heidelberg

*4 Institute of Neuropathology, Univ. Hosp. Giessen und

Marburg GmbH *5

Neurological Institute, Frankfurt University Medical School

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From left to right

First row: Dr. Ichidai Tanaka, Dr. Hiromi Furuta, Dr. Yoshitaka Sekido, Ms. Mari Kizuki, Dr. Makiko Fujii, Dr. Yuko Murakami-Tonami..

Second row: Dr. Asuki Fukatsu, Mr. Yoshio Tatematsu, Dr. Akihiro Matsushita, Dr. Hirotaka Osada, Ms. Yumi Nakahama, Mr. Ryuma Takao, Ms. Miwako Nishizawa, Dr. Shuhei Hakiri.

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Division of Molecular Oncology ________________________________________________________________________________ Yoshitaka Sekido M.D., Ph.D., Chief Hirotaka Osada, M.D., Ph.D., Section Head Yutaka Kondo, M.D., Ph.D., Section Head (until October 2012) Makiko Fujii, D.D.S., PhD., Senior Researcher Yuko Murakami-Tonami, M.D., Ph.D., Senior Researcher (as of September 2012) Yoshio Tatematsu, B.S., Research Assistant Yasue Matsudaira, B.S., Research Assistant (until March 2012) Miwako Nishizawa, B.P., Research Assistant (as of April 2012) Mari Kizuki, Semi-regular Employee (as of July 2012) Yumi Nakahama, Semi-regular Employee (as of March 2013) Research Resident Futoshi Ishiguro, M.D., Nagoya University Graduate School of Medicine (until March 2012) Yukiko Atsuta, M.D., Nagoya University Graduate School of Medicine (April ~ June 2012) Ichidai Tanaka, M.D., Nagoya University Graduate School of Medicine (July ~ December 2012), Research Resident of the Foundation for Promotion of Cancer Research (as of January 2013) Asuki Fukatsu, M.D., Nagoya University Graduate School of Medicine (as of April 2013) Fumiharu Ohka, M.D., Nagoya University Graduate School of Medicine (until October 2012) Keisuke Katsushima, M.S., Hokkaido University Graduate School of Life Science (April ~ October 2012) Visiting Trainees Keiko Shinjo, M.D., Nagoya University Graduate School of Medicine (until May 2012) Keisuke Katsushima, M.S., Hokkaido University Graduate School of Life Science (until March, 2012) Ichidai Tanaka, M.D., Nagoya University Graduate School of Medicine (April ~ June 2012) Asuki Fukatsu, M.D., Nagoya University Graduate School of Medicine (until March 2013) Shuhei Hakiri, M.D., Nagoya University Graduate School of Medicine (as of April 2013) Hiromi Furuta, M.D., Nagoya City University Graduate School of Medicine (as of April 2013) Akihiro Matsushita, M.D., Nagoya University Graduate School of Medicine (as of November 2013) Ryuma Takao, Meijo University (February ~ December 2013) Shiho Inagaki, Kinjo University (August ~November 2013) General Summary Our goal is to determine genetic lesions and epigenetic alterations giving rise to human solid cancers and use this information for prevention, diagnosis, and treatment of disease. The research also provides opportunities to dissect biochemical and pathological pathways of malignant phenotypes including deregulated cell proliferation, invasion, metastasis and drug resistance. Currently, we are especially focusing on malignant mesothelioma and lung cancer. Human cancers arise because of genetic mutations in oncogenes and tumor suppressor genes, and we are approaching this by study of candidate genes, systematic molecular analysis of biochemical pathways, and global approaches such as microarray analysis of gene expression profiling, comparative genomic hybridization techniques for chromosomal abnormalities, and next generation sequencing. Epigenetic changes with DNA methylation, histone modification, and microRNA expression also indicate these to be not only involved in inactivation of tumor suppressor genes but also to contribute to fundamental mechanisms of regulation of maintenance of cancer stem cell populations and differentiated cell lineages . We also functionally analyze candidate genes by transducing wild type copies into human cancer cells and testing for their ability to suppress malignancy in vitro and in vivo as well as characterizing their protein products biochemically. Alternatively, we inactivate expression using RNA interference (RNAi) in either tumor or normal cells and then study resultant phenotypes. Understanding the functions of mutated genes and disrupted signaling pathways will hopefully provide a foundation for translational research for human malignancies from bench to bedside.

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1. LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via the Hippo signaling cascade

Tanaka, I., Osada, H., Fujii, M., Fukatsu, A., Hida, T.*1, Horio, Y.*1, Kondo, Y.*2, Sato, A.*3, Hasegawa, Y.*4, Tsujimura, T.*3, and Y Sekido

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to neurofibromatosis type 2 (NF2)–Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the detailed mechanisms of disordered YAP activation in MMs have yet to be well clarified. We investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently show downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor 2 (LATS2), one of the last-step kinases in the NF2-Hippo pathway. Although loss of AJUBA expression proved independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We further found that AJUBA transduction into MM cells significantly suppressed promoter activity of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of MM cells featuring normal LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation. *1Department of Thoracic Oncology, Aichi Cancer Center, Nagoya, Japan *2Division of Epigenomics, Aichi Cancer Center Research Institute, Nagoya, Japan *3Department of Pathology, Hyogo College of Medicine, Nishinomiya, Japan *4Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan;

2. RASSF3 downregulation increases

malignant phenotypes of non-small cell lung cancer

Fukatsu, A., Ishiguro, F.*1, Tanaka, I., Kudo, T*2, *3, Nakagawa, K.*2, Shinjo, K.*4, Kondo, Y.*5, Fujii, M., Hasegawa, Y.*6, Tomizawa, K.*7, Mitsudomi, T.*7, *8, Osada, H., Hata, Y.*2, and Sekido, Y.

Ras-Association Family1A (RASSF1A) is a well-established tumor suppressor. Ten RASSF homologues comprise the family, and each member is considered a tumor suppressor. RASSF3 is one for which functions have yet to be clarified in detail. Recently, we found that RASSF3 interacts with MDM2 and facilitates its ubiquitination, which induces apoptosis through p53 stabilization.

Fig. 1 (A) Western blotting analysis of AJUBA, YAP and phospho-YAP Ser127 (p-YAP). AJUBA expression was undetectable in 15 and significantly low in 3 of 24 MM cell lines compared to an immortalized normal mesothelial cell line, MeT-5A. YAP phosphorylation was remarkably reduced in 21 of 24 MM cell lines compared to MeT-5A. Expression of β-actin used as a control. (B) Cell proliferation assays. After infection with AJUBA-expressing or empty lentiviruses, calorimetric assays were performed at each time point. AJUBA transduction significantly inhibited the cell proliferation of NCI-H290 and Y-MESO-8D cells. (C) Soft agar colony formation assays with AJUBA transduction. After 12-days incubation, colonies were stained with 0.03% crystal violet. Anchorage-independent growth was significantly suppressed in NCI-H290 and Y-MESO-8D cell lines.

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However, the role of RASSF3 in human malignancies remains largely unknown. Ninety-five non-small cell lung cancer (NSCLC) patients from Nagoya University Hospital and 45 NSCLC patients from Aichi Cancer Center Hospital underwent pulmonary resection at each hospital, and lung cancer and corresponding non-cancerous lung tissues were collected. The expression levels of RASSF3 were analyzed using quantitative real-time reverse transcription PCR. We performed statistical analysis to investigate the correlation with RASSF3 expression and clinicopathological characteristics. We also transfected RASSF3-siRNA into NSCLC cells, and performed motility assays to evaluate the influence on migration ability. RASSF3 expression levels were down-regulated in 125 of the total of 140 NSCLCs. On multivariate logistic regression analysis, low RASSF3 expression below the median value was independently correlated with progressive phenotypes (lymph node metastasis and pleural invasion), non-adenocarcinoma histology and wild-type epidermal growth factor receptor (EGFR) status. In motility assays, RASSF3-knockdown NSCLC cells increased the migration rate compared to control cells. Together, our findings indicate RASSF3 is a candidate tumor suppressor gene for NSCLCs. *1Departments of General Thoracic Surgery, Graduate School of Medicine, Nagoya University, Nagoya, Japan *2Departments of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan *3Departments of Neurosurgery, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan *4Departments of Oncological Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan *5Division of Epigenomics, Aichi Cancer Center Research Institute, Nagoya, Japan *6Departments of Respiratory Medicine, Graduate School of Medicine, Nagoya University, Nagoya, Japan *7Department of Thoracic Surgery, Aichi Cancer Center Hospital, Nagoya, Japan *8Department of Thoracic Surgery, Kinki University Faculty of Medicine, Sayama, Japan

Fig. 2. (A) Relative expression levels of the RASSF3 gene of NSCLC tumor samples from Nagoya University hospital and corresponding non-cancerous lung tissues. The mean RASSF3 expression level of the non-tumor samples was arbitrarily set as 1.0. Expression levels of tumor (blue line) and lung tissue (red line) are indicated side by side from the same patients, with the lowest RASSF3 level of the tumor starting from the left. Note the frequent down-regulation in NSCLCs. (B) Wound healing assay of RASSF3-knockdown cells. Forty-eight hours after transfection, A549 cells were re-plated in 3.5 cm dishes. After the cells had grown to confluence, cultures were damaged using a 1-200 μl beveled orifice tip and then allowed to migrate. Photographs were taken at the indicated time points. RASSF3-knockdown cells demonstrated increased migration rates as compared to the control. siCont; siRNA of negative control, siRF3; siRNA against RASSF3.

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From left to right

First row: Dr. S. Tsuzuki, Dr. M. Seto, Ms. K. Hirano, Ms. Y. Kasugai Second row: Ms. S. Sato, Dr. N. Yoshida, Dr. T. Takahara, Dr. M. Katayama, Dr. T. Kakiuchi

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Division of Molecular Medicine ________________________________________________________________________________ Masao Seto, M.D., Ph.D. Chief Shinobu Tsuzuki, M.D., Ph.D. Section Head Kennosuke Karube, M.D., Ph.D. Senior Researcher (until December 2012) Miyuki Katayama, M.D., Ph.D. Senior Researcher Yumiko Kasugai, B.S. Research Assistant Kotaro Arita, M.D. Research Resident (until October 2013) Taishi Takahara, M.D. Research Resident Kyoko Hirano, B.S. Research Assistant Visiting Trainees Noriaki Yoshida, M.D. Nagoya University Graduate Shool of Medicine Tatsuo Kakiuchi, M.D. Nagoya University Graduate Shool of Medicine General Summary Research in this laboratory is focused on elucidating genetic and molecular bases of human cancer, with a view to applying the obtained knowledge for clinical oncology. We are working on hematological malignancies, in collaboration with physicians in the Department of Hematology and Cell Therapy (Chief, Dr. Tomohiro Kinoshita) of Aichi Cancer Center Central Hospital. The group of diseases is highly associated with genetic changes so that some malignancies can be classified according to genetic changes specific to given entities. Such close association provides evidence that the genetic changes play pivotal roles in disease development and/or clinicopathological manifestations. Over the last two years, we have studied several issues in particular: (1) possible involvement of deregulated cell cycling, in particular that associated with loss of CDKN2A, in the transformation of chronic type adult T-cell leukemia/lymphoma and in its clinical outcome; (2) establishment of a method to generate lymphomas in mice employing in vitro-induced germinal center B and T cells; (3) identification of effective drugs against NK cell lymphoma; (4) involvement of clonal heterogeneity in the development of lymphoma and its impact on clinical outcome; (5) modeling first-hit functions of the TEL-AML1 fusion gene in establishment of a pre-leukemic state. 1. Cell cycle deregulation contributes to

acute transformation in chronic type adult T-cell leukemia/lymphoma

Yoshida, N., Karube, K., Utsunomiya, A.*1, Tsukasaki,

K.*2, Imaizumi, Y.*

2, Taira, N.*

3, Uike, N.*

4, Umino, A.,

Arita, K., Katayama, M., Tsuzuki, S., Kinoshita, T.*5,

Ohshima, K.*6, and Seto, M.

Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1 induced neoplasm with four clinical subtypes: acute, lymphoma, chronic, and smoldering. Although chronic and smoldering subtypes are regarded as indolent, about half of these cases progress to acute type ATL and subsequent death. Therefore, acute transformation is a predictive indicator for patients with indolent ATL. However, the underlying molecular mechanisms have not fully elucidated. In the present study, oligo-array comparative genomic hybridization (aCGH) and comprehensive gene-expression profiling (GEP) were applied to 27 and 35 cases of chronic and acute type ATL, respectively, in an effort to determine molecular

pathogeneses, and especially the molecular mechanisms of acute transformation. The use of aCGH revealed that genomic loss of 9p21.3 was significantly characteristic of acute type ATL, but not of chronic type ATL. Among the genes located in the loss region, only CDKN2A expression was reduced in accordance with genomic loss. Re-expression of CDKN2A (and especially of INK4a) suppressed proliferation of ATL cell lines. Clinically, chronic type ATL patients with alterations in cell cycle-related genes including CDKN2A showed a poorer prognosis than patients without such alterations. Additionally, the results of GEP indicated that the pathophysiology of chronic type ATL samples with such alterations is close to that of acute type ATL. These findings suggested that cell cycle-related genes play an important role in acute transformation and should serve as good new prognostic markers for chronic type ATL.

*1 Department of Hematology, Imamura Bun-in Hospital, Kagoshima, Japan *2 Department of Hematology, Atomic Bomb Disease

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and Hibakusha Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan *3 Department of Internal Medicine, Heartlife Hospital, Okinawa, Japan *4 Department of Hematology, National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan *5 Department of Hematology and Cell Therapy, Aichi Cancer Center, Nagoya, Japan *6 Department of Pathology, School of Medicine, Kurume University,Kurume, Japan

2. Generation of mouse models of

lymphoid neoplasms using retroviral gene transduction of in vitro-induced germinal center B and T cells

Arita, K., Maeda-Kasugai, Y., Ohshima, K.*1, Tsuzuki, S.,

Katayama, M., Karube, K., Yoshida, N., Sugiyama, T.*2,

Seto, M. Evidence is accumulating that hematologic

malignancies develop following acquisition of multiple genetic changes. Despite providing many insights into the way by which given genetic changes contribute to the development of disease, the generation of animal models is often laborious. We have focused on a simplified method that allows the retroviral transduction of genes of interest into mouse B or T cells, thus leading to rapid generation of models of lymphoid neoplasms in mice. Specifically, germinal center B cells induced in vitro from naive mouse B cells and infected with retroviruses for Myc and Bcl2 rapidly developed into neoplasms of immunoglobulin-expressing mature B cells in transplanted mice. Likewise, T cells induced in vitro from immature hematopoietic cells and infected with retroviruses for Myc, Bcl2, and Ccnd1 rapidly developed CD4(+)CD8(-) and CD4(+)CD8(+) T cell neoplasms after transplantation. These findings support use of our simplified method as a versatile tool for lymphoma research. *1 Department of Pathology, School of Medicine, Kurume University,Kurume, Japan *2 Third Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan

3. Comprehensive gene expression profiles of NK cell neoplasms indicate vorinostat as an effective drug candidate

Karube, K., Tsuzuki, S., Yoshida, N., Arita, K., Kato, H.*

1, Katayama, M., Ko, YH.*

2 , Ohshima, K.*

3,

Nakamura, S.*4, Kinoshita, T.*

1, Seto, M.

NK cell neoplasms are lymphoid malignancies with an aggressive clinical course. In the present

study, we analyzed gene expression profiles of NK cell neoplasms and attempted to identify important molecular pathways and new effective drugs. Pathway analysis of gene expression profiles suggested important roles of the JAK-STAT, NF-κB or Wnt pathways. Notably, Western blot analysis revealed that STAT3 was expressed and phosphorylated at a higher level in NK cell lines than in normal NK cells or other cell lines, indicating the occurrence of JAK-STAT activation in NK cell neoplasms. Connectivity Map (CMAP) analysis of gene expression profiles identified candidate drugs against NK cell neoplasms. Among the suggested drugs , we focused on puromycin, phenoxybenzamine, LY294002, wortmannin, vorinostat and trichostatin A because they exhibited high enrichment scores. When added to NK cell lines and other cell lines, among the drugs, vorinostat suppressed NK cell line proliferation at a significantly lower concentration. Suppression of the JAK-STAT pathway appeared to contribute to this effect. Vorinostat may be a good candidate for use in therapy against NK cell neoplasms.

*1 Department of Hematology and Cell Therapy, Aichi Cancer Center Hospital *2 Department of Pathology, Samsung Medical Sungkyunkwan University School of Medicine, Republic of Korea *3 Department of Pathology, School of Medicine, Kurume University, Kurume, Japan *4 Department of Pathology and Clinical Laboratories, Nagoya University Hospital, Nagoya, Japan

4. Clonal heterogeneity of lymphoid

malignancies is related to poor prognosis

Katayama, M., Tagawa, H.*1, Nakagawa, M.*2, Fukuhara, N.*3, Sivasundaram, S.*4, Takeuchi, I. *5, Kasugai, Y., Arita, K., Yoshida, N., Kakiuchi, T., Karube, K., Tsuzuki. S, Nakamura, S.*6, Seto, M.

Clonal heterogeneity means several sub-clones with different genomic aberrations in any particular tumor sample. Most of the cases with clonal heterogeneity result from clonal evolution which makes tumors more aggressive. Here we identified 332 lymphoma samples (31 MALT lymphomas, 24 Burkitt lymphomas, 29 mantle cell lymphomas, 79 follicular lymphomas, 118 diffuse large B-cell lymphomas (DLBCLs) and 51 peripheral T-cell lymphomas, not otherwise specified) with or without clonal heterogeneity using array CGH analysis. The frequency of clonal heterogeneity for each lymphoma varied from 24% to 69%. Genomic aberrations for samples with clonal heterogeneity in multiple lymphoma species

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included loss/deletion of 9p21.3 (CDKN2A/2B locus) and 17p13 (TP53, ATP1B2, SAT2, SHBG locus). DLBCLs and mantle cell lymphomas with clonal heterogeneity demonstrated a significantly poorer prognosis than samples without clonal heterogeneity (p = 0.005 and p = 0.008, respectively). These results that clonal heterogeneity involving common genomic aberrations is present in all lymphoma types and is related to a poor prognosis, indicating this common phenomenon to be important for tumor progression.

*1 Department of Hematology, Nephrology, and Rheumatology, Akita University Graduate School of Medicine, Akita, Japan *2 Metabolism Branch, Center for Cancer Research, National Cancer Institute, USA *3 Department of Hematology and Rheumatology, Tohoku University Hospital, Sendai, Japan *4 Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Japan *5 Department of Computer Science/Scientific and Engineering Simulation, Nagoya Institute of Technology, Nagoya, Japan *6 Department of Pathology and Clinical Laboratories, Nagoya University Hospital, Nagoya, Japan

5. TEL (ETV6)-AML1 (RUNX1) initiates

self-renewing fetal pro-B cells in association with a transcriptional program shared with embryonic stem cells in mice

Tsuzuki, S., and Seto, M. The initial steps involved in the pathogenesis of

acute leukemia are poorly understood. TEL-AML1 fusion gene usually arises before birth, producing a persistent and covert preleukemic clone that may convert to precursor B cell leukemia following accumulation of secondary genetic ‘‘hits.’’ Here, we showed that TEL-AML1 can induce persistent self-renewing pro-B cells in mice. TEL-AML11 cells nevertheless differentiate terminally in the long term, providing a ‘‘window’’ period that may allow secondary genetic hits to accumulate and lead to leukemia. TEL-AML1-mediated self-renewal is associated with a transcriptional program shared with embryonic stem cells (ESCs), within which Mybl2, Tgif2, Pim2, and Hmgb3 are critical components, sufficient to establish self-renewing pro-B cells. We further showed that TEL-AML1 increases the number of leukemia-initiating cells that are generated in cooperation with additional genetic hits, thus providing an overall basis for the development of novel therapeutic and preventive measures targeting the TEL-AML1-associated

transcriptional program.

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From left to right

First row: Dr. R. Zhang, Dr. A. Demachi-Okamura, Dr. K. Kuzushima, Ms. K. Hiramatsu Second row: Ms M. Tatsumi, Dr. N. Ueda Ms. K. Shiraishi, Dr. E. Yamada, Ms. R. Terada, Mr. H.

Maki Inset: Dr. Y. Akatsuka, Dr. Y Uemura, Ms. T. Tsuboi

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Division of Immunology ________________________________________________________________________________ Kiyotaka Kuzushima, M.D. Chief Mitsugu Fujita, M.D. Section Head (until March 2012) Yasushi Uemura, D.D.S. Senior Researcher (until August 2013) Ayako Demachi-Okamura, Ph.D. Researcher Shinji Kondo, M.D. Research Resident (until March 2012) Rong Zhang, Ph.D. Research Resident Eri Yamada, M.D. Research Resident (as of April 2012) Miwako Nishizawa, B.P. Senior Research Assistant (until March 2012) Kaho Hiramatsu, Research Assistant (as of April 2012) Rie Terada (Miura), Semi-regular Employee Minako Tatsumi, Semi-regular Employee Keiko Shiraishi, Semi-regular Employee Tomiko Tsuboi, Semi-regular Employee Visiting Researcher Yoshiki Akatsuka, M.D. Department of Hematology & Oncology, Fujita Health University Mitsugu Fujita, M.D. Department of Neurosurgery and Department of Microbiology, Kinki University (as of April 2012) Yasushi Uemura, D.D.S. Division of Cancer Immunotherapy, Exploratory Oncology Research & Clinical Trial Center (as of September 2013) Visiting Trainees Hiroyuki Maki, Department of Cellular Oncology, Nagoya University Graduate School of Medicine Eri Yamada, M.D. Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine (as of January 2012 until March 2012) Shinji Kondo, M.D. Department of Gynecologic Oncology, Aichi Cancer Center (as of April 2012) Norihiro Ueda, M.D. Department of Hematology and Oncology, Nagoya University Graduate School of Medicine (as of October 2012) General Summary The object of our research is to establish molecular and cellular bases for novel cancer therapy taking advantage of immune responses. The achievements during past two years are as follows.

Firstly, we explored the association of oncogenic K-ras mutation (G12V) with presentation of a CTL epitope derived from puromycin-sensitive aminopeptidase (PSA). Most pancreatic cancers are reported to harbor the mutation and consequently activated autophagy. In addition, the epitope has been demonstrated to be processed and presented in an autophagy-dependent manner. After transfection of the mutated K-ras and HLA-A*24:02, non-malignant human epithelial cells presented the epitope accompanied by induction of autophagy. These data suggest that the constitutive autophagy evoked by K-ras mutation causes unique processing and presentation of the CTL epitope, providing basic information for immunotherapy.

Secondly, we sought to generate highly specific monoclonal antibodies (mAbs) for a minor histocompatibility antigen (mHAg), HA-1H, by immunizing HLA- A*02:01-transgenic mice with tetramerized recombinant HLA-A*02:01 protein incorporating the HA-1H peptide and β2-microglobulin (HA-1H/HLA-A2). The phage display technique was applied to select clones reactive to HA-1H/HLA-A2. Details are described in the text below.

Thirdly, we established a system to facilitate proliferation of embryonic stem (ES) or induced pluripotent stem (iPS) cell-derived myeloid cells. The cells exhibit cytokine-dependent proliferation and dendritic cell (DC)-like differentiation and thus may offer a solution to current problems in preparing sufficient numbers of DCs. Lastly, we investigated the role of human invariant natural killer (iNK) T cells in regulation of IL-12p70 and osteopontine (OPN) production by DCs. We found that iNKT cells modify the IL-12p70/OPN balance to

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enhance IL-12p70-induced cell-mediated immunity and suppress the OPN-dependent inflammatory condition that promotes tumor development. An aspect of the protective role of iNKT cells in tumor immunosurveillance seems to be elucidated by these findings. 1. Constitutively active autophagy causes

a CTL epitope generation in pancreatic cancer cells

Demachi-Okamura, A., Kuzushima, K. Tumor antigen-specific cytotoxic T

lymphocytes (CTLs) play a crucial role in immune system-mediated elimination of cancer. They exert such effects through recognition of tumor antigen-derived peptides bound to human leukocyte antigens (HLAs) on cell surfaces. Antigen processing pathways to produce the peptides in particular cancer cells remain to be fully elucidated. To study one such pathway, we used a CTL clone designated 16F3 which recognizes puromycin-sensitive aminopeptidase (PSA) in an HLA-A*24:02-restricted manner. Generation of the epitope recognized by 16F3 requires proteasomal degradation and transportation from the endoplasmic reticulum to the Golgi apparatus as demonstrated in drug inhibition assays.

Autophagy is also required in the epitope processing as evidenced by RNAi-mediated autophagy-mediated gene silencing. Immunofluorescence revealed high levels of autophagic activity in pancreatic cancer cells that are sensitive to CTL-mediated lysis. As previously reported, constitutively activated autophagy is associated with oncogenic K-ras mutation (G12V) in such pancreatic cancer cells. Thus we sought to study whether K-ras mutation mightresult in PSA epitope generation through activation of autophagy.

To this end, MCF10A, a non-tumorigenic human breast epithelial cell line, was transfected with mutated K-ras. Many autophagosomes emerged in MCF10A cells after the transfection. Additional transfection of HLA-A*24:02 enabled the cells to stimulate 16F3 for IFN-γ production. Interestingly, induction of autophagy using chemicals or nutritional deprivation did not result in the same epitope liberation as observed with K-ras mutation. These data suggest that the constitutively active autophagy evoked by K-ras mutation causes unique processing and presentation of the CTL epitope in a part of pancreatic cancer cells and provide important information for cancer immunotherapy.

2. Construction and molecular character- ization of a T-cell receptor-like antibody and CAR-T cells specific for

minor histocompatibility antigen HA-1H Akatsuka, Y.*1,2, Inaguma, Y.*1, Akahori, Y.*3, Murayama, Y.*1, Shiraishi, K., Endoh, A.*1, Demachi-Okamura, A., Hiramatsu, K., Nishimura, Y.*4, Takahashi, To.*5, Emi, N.*1, and Kuzushima, K.

Selective graft-versus-tumor (GVT) reactivity with minimal risk of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation is thought to be induced by targeting minor histocompatibility (H) antigens (Ags) expressed only on patients’ hematopoietic cells. Among HLA-A*02:01 positive patients, minor H Ags such as HA-1 have been shown to be associated with anti-tumor responses with minimal GVHD and thus have been explored for applications in adoptive immunotherapy. Because preparation of Ag-specific cytotoxic T cell clones (CTLs) or lines for adoptive immunotherapy is labor-intensive, genetic transfer of T-cell receptors (TCRs) directed toward target Ags into T lymphocytes has been employed to efficiently generate anti-tumor T cells. Alternatively, T cells could be gene-modified with a chimeric antigen receptor (CAR) harnessing a single chain antibody moiety (scFv). The conventional CAR strategy has the limitation of only targeting cell surface Ags on target cells. One possible way to attain intracellular Ag targeting with a CAR is to generate a TCR-like monoclonal antibody (mAb) as a source of scFv. We sought to generate highly specific mAbs specific for HA-1H minor H Ag by immunizing mice with tetramerized recombinant HLA-A2 incorporating HA-1H minor H Ag peptides and β2-microglobulin (HA-1H/HLA-A2). We hypothesized that the use of HLA-A2 transgenic mice, which should be tolerant to human HLA-A2, would facilitate efficient induction of mAbs specific for peptides presented on HLA-A2. Phage libraries were generated from splenic B cells and screened by panning for clones reactive to plate-bound HA-1H/HLA-A2 in the presence of irrelevant free MAGEA4/HLA-A2 for competition. Candidate scFv encoded by obtained phage clones was transformed to an scFv tetrameric Ab form or introduced into T cells as CAR coupled to CD28 transmembrane and CD3ζ domains (CD28-ζ). A total of 144 clones were randomly selected from 8.1×108 clones that had been recovered after the third panning. Among these, 18 (12.5%) showed preferential binding to HA-1/HLA-A2, 137 showed similar binding to both pMHC complexes, and 7

27

showed reactivity to neither of them. One of 18 scFv Abs, clone #131, demonstrated high affinity (KD = 14.9nM) for the HA-1H/HLA-A2 complex. Primary human T cells transduced with #131 scFv-CD28-ζ were stained with HA-1H/HLA-A2 tetramers as strongly as a cognate CTL clone, EH6, specific for endogenously HLA-A2- and HA-1H-positive cells. Unexpectedly, however, #131 scFv-CD28-ζ CAR-T cells required an ~100-fold higher Ag density when pulsed exogenously to exert cytotoxicity than did the cognate EH6-CTL. In addition, mAb blocking experiments demonstrated that #131 scFv-CD28-ζ CD8+ CAR-T cells were less sensitive to CD8 blockade when they were completely blocked with HA-1H/HLA-A2 tetramers, and even #131 scFv-CD28-ζ CD4+ CAR-T cells lysed the target in a similar way to #131 scFv-CD28-ζ CD8+ CAR-T cells. These data suggest that T cells with higher affinity antigen receptors than TCRs (average KD ranging between 1μM~100μM) are less able to recognize low density peptide/MHC antigens, as reported in the case of affinity-matured TCR or CAR, and that CD8+ CAR-T cells may not be necessarily CD8-dependent possibly due to failure to form complexes with CD3. Because #131 scFv-CD28-ζ CD8+ CAR-T cells could mount IFN-γ, TNF-α and IL-2 production when stimulated by K562/HLA-A2 cells transduced with HA-1H minigene, a different level of signaling threshold might exist between cytotoxic function and cytokine production. CAR-T cells with scFv with lower KD are currently being tested to examine the underlying mechanisms. *1 Department of Hematology, School of Medicine, Fujita Health University. *2 Visiting Researcher, Division of Immunology

*3 Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University. *4 Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University. *5 Aichi Comprehensive Health Science Center, Aichi Health Promotion Foundation.

3. Pluripotent stem cell-derived prolifera- ting myeloid cells as a possible basis for cellular vaccine cancer immuno- therapy

Zhang, R., Liu, T.*1, Maki, H., Kuzushima, K., and Uemura, Y.

Dendritic cells (DCs) are one of the attractive targets in efforts to generate therapeutic immunity against cancer, because of their ability to initiate

tumor-associated antigen (TAA)-specific T cell responses. With U.S. Food and Drug Administration approval of the first DC-based vaccine, Provenge, an autologous DC-based therapy is being established as a new cell modality for cancer treatment. However, the preparation of autologous DC therapeutics is expensive for patients and technically demanding for clinicians, not to mention the difficulty involved with large-scale industrial production.

Pluripotent stem cells such as embryonic stem (ES) cells and the recently developed induced pluripotent stem (iPS) cells have the potential to propagate indefinitely and possess the ability to differentiate into various types of somatic cells. Several groups, including ours, have thus far established methods to generate DCs from pluripotent stem cells and demonstrated utility of pluripotent stem cell-derived DC in cancer immunotherapy. However, the methods are too laborious for practical application in the clinic. In addition, generating DCs from pluripotent stem cells takes more than one month.

For the purpose of overcoming these obstacles, we established a system to induce proliferation of the ES or iPS cell-derived myeloid cells by lentivirus-mediated transduction of the cMYC gene. Proliferating myeloid cells (pMCs) have the ability to propagate for more than three months in a GM-CSF-dependent manner while retaining the potential to differentiate into DC-like cells. In maintenance culture medium containing both GM-CSF and M-CSF, the pMCs expressed DEC205, CD11b, CD11c, CD80, CD86, and Gr-1, but neither MHC-I nor MHC-II. Two days after removal of M-CSF, the pMCs differentiated into immature DC-like cells (pMC-DCs), as indicated by the expression of MHC-I and low levels of CD40, CD86, and MHC-II. Comparable to BM-derived immature DCs, they stimulated a primary mixed lymphocyte reaction (MLR). In addition, LPS exposure induced production of TNF-α and IL-12p70 and significantly enhanced the expression of MHC-II and CD86, suggesting a typical DC maturation. Furthermore, the in vivo transfer of pMC-DCs pulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-specific cytotoxic T cells and elicited protection against challenge with OVA-expressing melanoma.

Myeloid cells exhibiting cytokine-dependent proliferation and DC-like differentiation may offer a feasible cellular vaccine approach for cancer immunotherapy. This would offer a solution to the problems currently faced in preparing sufficient numbers of DCs.

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*1 Cancer Center, Chinese PLA General Hospital., Beijing, China.

4. Modification of IL-12p70/osteopontin balance in dendritic cells by ligand activation of invariant NKT cells

Zhang, R., Liu, T.*1, Kuzushima, K., and Uemura, Y. Invariant NKT cells constitute a unique subset

of T lymphocytes characterized by expression of a semi-invariant TCR composed of a canonical invariant TCR α-chain (Vα14-Jα18 for mice and Vα24-Jα18 for humans) and a TCR β-chain using limited Vβ segments (Vβ8.2, Vβ7, Vβ2 in mice and a Vβ11 in humans). The biased TCR usage appears to confer on iNKT cells the capacity to recognize a limited number of glycolipid antigens presented by CD1d. α-Galactosylceramide (α-GalCer), a synthetic glycosphingolipid originally derived from a marine sponge, activates iNKT cells to rapidly produce large amounts of both Th1 and Th2 cytokines, which, in turn, license dendritic cell (DC) functions, promoting protective antitumor immunity. However, the nature of the protective role of invariant NKT (iNKT) cells in the tumor immunosurveillance remains to be fully elucidated.

IL-12p70, a heterodimeric cytokine comprised of a p35 subunit and a p40 subunit, plays a crucial role in the cell-mediated immune response. Contrasting with the function of IL-12p70, osteopontine (OPN) is a potent proinflammatory cytokine that can be expressed by both tumor cells and cellular components of the tumor microenvironment. Elevated levels of OPN in tumor tissue and blood circulation are associated with tumor metastasis and a poor prognosis.

We investigated the role of human iNKT cells in the regulation of IL-12p70 and OPN production by DCs. Activation of iNKT cells by their specific ligand α-GalCer enhanced IL-12p70 while inhibiting the OPN production. The differential regulation of IL-12p70/OPN appears mainly mediated by their harmonious Th1/2 cytokine production. In particular, the down regulation of OPN was found to be associated with a production of IL-4, IL-13, and IFN-γ from iNKT cells. In contrast, these cytokines synergistically enhanced IL-12p70 production. These findings indicate that iNKT cells modify the IL-12p70/OPN balance to enhance IL-12p70-induced cell-mediated immunity and suppress the OPN-dependent inflammatory condition that promotes tumor development. *1 Cancer Center, Chinese PLA General Hospital.,

Beijing, China.

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From left to right

Dr. Teru Kanda, Dr. Daisuke Kawashima, Dr. Sho Nakasu, Ms. Ayako Watanabe, Mr. Yohei Narita, Dr. Tatsuya Tsurumi

30

Division of Virology ________________________________________________________________________________ Tatsuya Tsurumi, M.D., Ph.D. Chief Teru Kanda, M.D., Ph.D. Section Head Sho Nakasu, PhD. Senior Researcher Takayuki Murata, Vet. M.D., Ph.D. Researcher (until April 2013) Shinichi Saito, Ph.D. Research Resident (until August 2012) Daisuke Kawashima, Ph.D. Research Resident Chieko Noda, Research Assistant (until March 2012) Yohei Narita, Research Assistant (as of April 2012) Toshiko Gamano, Semi-regular employee (until May 2013) Visiting Trainee Atsuko Sugimoto, Nagoya University Graduate School of Medicine Yohei Narita, Nagoya University Graduate School of Medicine Yoshitaka Sato, Kobe University School of Medicine General Summary Approximately 15% of all human cancers have a viral etiology, but only six viruses have actually been unequivocally implicated in neoplastic development. Among these the Epstein-Barr virus (EBV) is the primary object of our own studies. EBV is a ubiquitous gamma herpesvirus associated with several malignant diseases, including Burkitt’s lymphoma, nasopharyngeal lymphoma, a subset of Hodgkin’s lymphomas, some gastric cancers, and B cell lymphomas in immunosuppressed patients. Our research aims are to elucidate the molecular mechanisms of viral proliferation and oncogenesis of EBV as part of the world-wide effort to combat virus-infected cancers. During the period 2012-2013, our research interest was concentrated on the following issues: 1) Interaction between basic residues of Epstein-Barr virus EBNA1 protein and cellular chromatin mediates viral plasmid maintenance; 2) Contributions of myocyte enhancer factor 2 (MEF2) family transcription factors to BZLF1 expression in Epstein-Barr virus reactivation from latency; 3) Repression by heat shock protein 90 inhibitors of latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma cells; 4) Different distributions of Epstein-Barr virus early and late gene transcripts within viral replication compartments; 5) Nuclear transport of Epstein-Barr virus DNA polymerase dependence on the BMRF1 polymerase processivity factor and molecular chaperone Hsp90; 6) Epstein-Barr virus deubiquitinase downregulation of TRAF6-mediated NF-κB signaling during productive replication; 7) Pin1 interaction with the Epstein-Barr virus DNA polymerase catalytic subunit and regulation of viral DNA replication; 8) HLA-restricted presentation of WT1 tumor antigens in B-lymphoblastoid cell lines established using a maxi-EBV system; 9) Epigenetic histone modification of the Epstein-Barr virus BZLF1 promoter during latency and reactivation in Raji cells. 1. Interaction between Basic Residues of

Epstein-Barr Virus EBNA1 Protein and Cellular Chromatin Mediates Viral Plasmid Maintenance

Kanda, T. and Tsurumi, T. The Epstein-Barr virus (EBV) genome is

episomally maintained in latently infected cells, the viral protein EBNA1 acting as a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes

(chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanisms by which the CBDs of EBNA1 attach to cellular chromatin are still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 (amino acids 40–54) and CBD2 (amino acids 328–377) regions with alanines progressively impaired chromosome binding of EBNA1. Complete arginine-to-alanine substitutions within the CBD1 and -2 regions abolished the

31

ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effects, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine-rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, whereas a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between basic amino acids within the CBDs and negatively charged cellular chromatin.

2. Contribution of Myocyte Enhancer

Factor 2 (MEF2) Family Transcription Factors to BZLF1 Expression in Epstein-Barr virus Reactivation from Latency

Murata, T. and Tsurumi, T. Reactivation of the Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp)

normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including MEF2B, KLF4, and some cellular b-zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we then prepared recombinant EBV-BAC in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293. The virus failed to induce a subset of early genes, such as BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could completely be lifted by exogenous supply of BRLF1 together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1 but also BRLF1, in comparison to wild-type or revertant virus cases. We also observed MEF2 mutant EBV to feature relatively high repressive histone methylation, such as H3K27me3, but CpG DNA methylation levels were comparable around Zp and Rp. These findings shed light on BZLF1 expression and EBV reactivation from latency. 3. Heat shock protein 90 inhibitors

repress latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma cells

Murata, T. and Tsurumi, T. Epstein-Barr virus (EBV) LMP1, a major

oncoprotein expressed in latent infection, functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 natural killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγnull (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Fig. 1. These live confocal microscopic images depict an mCherry (red fluorescent protein) fused to the chromosome binding domains of Epstein-Barr virus EBNA1 colocalizing with histone H2B-GFP-labeled chromatin in mitotic HeLa cells. A merged image is at the bottom. This imaging system was used to reveal that the basic nature of the chromosome binding domains of EBNA1 is primarily important for its chromosome binding.

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4. Different Distributions of Epstein-Barr Virus Early and Late Gene Transcripts within Viral Replication Compartments

Sugimoto, A., Sato, Y., Murata, T., Kanda, T., and Tsurumi, T.

Productive replication of the Epstein-Barr virus (EBV) occurs in discrete sites in nuclei, called replication compartments, where viral genome DNA synthesis and transcription take place. The replication compartments include subnuclear domains, designated BMRF1 cores, which are highly enriched in the BMRF1 protein. During viral lytic replication, newly synthesized viral DNA genomes are organized around and then stored inside BMRF1 cores. Here, we examined spatial distribution of viral early and late gene mRNAs within replication compartments using confocal laser scanning microscopy and three-dimensional surface reconstruction imaging. EBV early mRNAs were mainly located outside the BMRF1 cores, while viral late mRNAs were identified inside, corresponding well with the fact that late gene transcription is dependent on viral DNA replication. From these results, we speculate that sites for viral early and late gene transcription are separated with reference to BMRF1 cores.

5. Nuclear Transport of Epstein-Barr Virus DNA Polymerase is Dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90

Kawashima, D., Murata, T., Kanda, T., and Tsurumi T. Epstein-Barr virus (EBV) replication proteins are transported into the nucleus to synthesize viral genomes. We have focused on molecular mechanisms for nuclear transport of EBV DNA polymerase. The EBV DNA polymerase catalytic subunit BALF5 was found to accumulate in the cytoplasm when expressed alone, while the EBV DNA polymerase processivity factor BMRF1 moved into the nucleus by itself. Coexpression of both proteins, however, resulted in efficient nuclear transport of BALF5. Deletion of the nuclear localization signal of BMRF1 diminished the protein nuclear transport, although both proteins could still interact. These results suggest that BALF5 interacts with BMRF1 to effect transport into the nucleus. Interestingly, we found that Hsp90 inhibitors or knockdown of Hsp90β with short hairpin RNA prevented the BALF5 nuclear transport, even in the presence of BMRF1, both in transfection assays and in the context of lytic replication. Immunoprecipitation analyses

Fig. 2. RNAPII becomes localized inside BMRF1 cores at late stages of productive replication. Tet-BZLF1/B95-8 cells were treated with doxycycline to induce lytic replication and harvested at the indicated post-induction times. After treatment with mCSK buffer, they were fixed. (A) Harvested cells were stained with anti-BMRF1 (green), anti-BALF2 (red), and anti-RNAPII (yellow) antibodies and observed by laser scanning confocal microscopy. The 2D images show brightest-point projections. Lower panels are merged images of the indicated combinations of the proteins. Pol II, RNA polymerase II. (B) Lytic replication-induced cells were stained with anti-BMRF1 (green) and anti-RNAPII (red). Projections of 60 images collected at 0.33-μm steps in the z axis are displayed as 3D topographical reconstruction images of BMRF1 and RNAPII (left and middle panels, respectively). Representative 3D surface reconstruction images are presented. Right panels are merged 3D surface reconstruction images. (C) The ratio of cells in which RNAPII was located outside the BMRF1 core to total BMRF1-positive cells was determined (black bars). White bars show the ratios of cells in which RNAPII was located inside the BMRF core to total BMRF1-positive cells. We regarded cells as inside when 50% or more of RNAPII signals were detected inside the core. More than 100 cells were counted at each of the indicated time points.

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suggested that the molecular chaperone Hsp90 interacts with BALF5. Treatment with Hsp90 inhibitors blocked viral DNA replication almost completely during lytic infection, and knockdown of Hsp90β reduced viral genome synthesis. Collectively the results indicate that Hsp90 interacts with BALF5 in the cytoplasm to assist complex formation with BMRF1, leading to nuclear transport. Hsp90 inhibitors may be useful for therapy for EBV-associated diseases in the future. 6. Epstein-Barr Virus Deubiquitinase

Downregulates TRAF6-mediated NF-κB Signaling during Productive Replication

Saito, S., Murata, T., Kanda, T., and Tsurumi, T. Epstein-Barr virus (EBV), a human oncogenic

herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was found to be suppressed upon onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have established that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. Thus HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB

signal transduction, leading to promotion of viral lytic DNA replication. 7. Pin1 Interacts with the Epstein-Barr

Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

Narita, Y., Murata, T., Kanda, T., and Tsurumi, T. Peptidyl-prolyl cis-trans isomerase

NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication.

Fig. 3. A schematic model demonstrating inhibition of NF-κB signaling by BPLF1 in the EBV life cycle. In EBV latent infection, NF-κB is activated by viral LMP1 protein; TRAF6 associates with LMP1 and is constitutively polyubiquitinated. Activation of NF-κB confers cell survival as well as inhibiting spontaneous lytic replication. Changes in the host cell microenvironment or other unknown triggers can downregulate the NF-κB activity and disrupt the balance between the latent cycle and the lytic cycle of EBV. Once lytic replication is induced, BPLF1 then deubiquitinates and inactivates TRAF6 to further block NF-κB signaling, promoting efficient viral genome replication.

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8. HLA-restricted Presentation of WT1 Tumor Antigen in B-lymphoblastoid Cell Lines Established using a Maxi-EBV System

Kanda, T., Kuzushima, K.*1, and Tsurumi, T. Lymphoblastoid cell lines (LCLs), which are

established by in vitro infection of peripheral B-lymphocytes with Epstein-Barr virus (EBV), are effective antigen-presenting cells. However, the ability of LCLs to present transduced tumor antigens has not yet been evaluated in detail. We report a single-step strategy utilizing a recombinant EBV (maxi-EBV) to convert B-lymphocytes from any individuals into indefinitely growing LCLs expressing a transgene of interest. The strategy was successfully used to establish LCLs expressing Wilms' tumor gene 1 (WT1) tumor antigen (WT1-LCLs), which is an attractive target for cancer immunotherapy. The established WT1-LCLs expressed more abundant WT1 protein than K562 leukemic cells, which are known to overexpress WT1. A WT1-specific cytotoxic T lymphocyte line efficiently lysed the WT1-LCL in a human leukocyte antigen-restricted manner, but poorly lysed control LCL not expressing WT1. These results indicate that the transduced WT1 antigen is processed and presented on the WT1-LCL. This experimental strategy can be applied to establish LCLs expressing other tumor antigens and should find a broad range of applications in the field of cancer immunotherapy. 9. Epigenetic Histone Modification of the

Epstein-Barr Virus BZLF1 Promoter during Latency and Reactivation in Raji Cells

Murata, T., Kondo, Y.*2, and Tsurumi, T. The Epstein-Barr virus (EBV) predominantly

establishes latent infection in B cells, and the

reactivation of the virus from latency is dependent on the expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores, or histone deacetylase (HDAC) inhibitors. In some cell lines latently infected with EBV, an HDAC inhibitor alone can induce BZLF1 transcription, while the treatment does not enhance expression in other cell lines, such as B95-8 or Raji cells, suggesting unknown suppressive mechanisms besides histone deacetylation in those cells. Here, we found epigenetic modification of the BZLF1 promoter in latent Raji cells by histone H3 lysine 27 trimethylation (H3K27me3), H3K9me2/me3, and H4K20me3. Levels of active markers such as histone acetylation and H3K4me3 were low in latent cells but increased upon reactivation. Treatment with 3-deazaneplanocin A (DZNep), an inhibitor of H3K27me3 and H4K20me3, significantly enhanced the BZLF1 transcription in Raji cells when in combination with an HDAC inhibitor, trichostatin A (TSA). Knockdown of Ezh2 or Suv420h1, histone methyltransferases for H3K27me3 or H4K20me3, respectively, further proved the suppression of Zp by methylation. Taken together, the results indicate that H3K27 methylation and H4K20 methylation are involved, at least partly, in the maintenance of latency, and histone acetylation and H3K4 methylation correlate with the reactivation of the virus in Raji cells. *1 Division of Immunology, Aichi Cancer Center Research Insutitute *2 Division of Epigenomics, Aichi Cancer Center Research Insutitute

Fig. 4. Summary of epigenetic histone modifications in the BZLF1 promoter of Raji cells. Repressive histone H3K9, H3K27, H4K20 methylation (marked with blue circles) is present in latency, and is not appreciably decreased even after induction. High levels of active markers, such as H3K4 methylation and histone acetylation (green circles), are notably associated with lytic induction.

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From left to right

First row: Dr. T. Fujishita, Dr. M. Aoki, Dr. R. Kajino, Ms. Y. Goto Second row: Dr. R. Maeda, Dr. K. Sakuma, Mr. M.V.Boxtel, Dr. Y. Kojima

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Division of Molecular Pathology ________________________________________________________________________________ Masahiro Aoki, M.D., D.M.Sc., Chief Keiichiro Sakuma, M.D., D.M.Sc., Senior Researcher Yasushi Kojima, M.D., D.M.Sc., Senior Researcher Rie Kajino., Ph.D. Researcher (as of April 2012) Teruaki Fujishita, D.M.Sc Researcher (as of April 2013) Yoshiko Goto, D.V.M.S., Research Assistant Ryo Maeda, M.D., Research Resident (as of April 2013) Visiting Scientists Reiji Kannagi, M.D., D.M.Sc. Visiting Trainees Sachiko Kondo, M.E., Nagoya City University (until March 2013) Mark van Boxtel, Radboud University Nijmegen (as of September 2013) General Summary

The incidence of colon cancer is increasing steadily in Japan; the disease is predicted to become the most common cancer as of 2020. Accumulating evidence suggests that in addition to genetic and epigenetic changes in the genome of cancer cells, interactions of cancer cells with non-cancer stromal cells play essential roles in support of colon cancer progression. Our research has aimed mainly to identify novel molecular targets for prevention and/or therapy of colon cancer through careful analyses of the intestinal tumor progression in genetically engineered mouse models, followed by evaluation of the findings using clinical samples. We are currently interested in the following subjects: (1) Genetic dissection of the signaling pathways that play pivotal roles in colon carcinogenesis; (2) Elucidating the complex tumor-stroma interactions that promote invasion and metastasis of colon cancer; (3) Identification of novel metastasis suppressor genes in colon cancer; and (4) Unraveling the pathophysiology of cancer cachexia. 1. c-Myc and CDX2 mediate E-selectin

ligand glycan expression in colon cancer cells undergoing EMT

Sakuma, K., Kannagi, R., and Aoki, M. Sialyl Lewis x (sLex) and sialyl Lewis a (sLea)

are E-selectin ligand glycans expressed on many types of cancer cells including colon cancer. Despite their clinical usefulness as tumor markers, supported by the accumulating evidence showing strong correlation between their expression levels and cancer progression, the mechanism underlying their expression remains elusive. In this study, we addressed a link between the sLex/a expression levels and epithelial-mesenchymal transition (EMT), a critical step during the progression of epithelial tumors.

We first determined by immunohistochemistry the sLex/a expression levels in human colon cancer cells undergoing EMT. The results indicated a correlation between the loss of cell-membranous E-cadherin and high sLea levels, supporting their link. We then addressed if there could be a common mechanism underlying sLex/a expression and EMT,

using HT29 and DLD-1 colon cancer cells. Treatment of the cells with EGF and/or bFGF in the absence of fetal bovine serum dramatically induced EMT and significantly increased the sLex/a expression levels. The transcript levels of ST3GAL1/3/4 and FUT3, encoding sialyltransferases and a fucosyltransferase, respectively, which directly promote sLex/a synthesis, were elevated upon EMT, while the transcript level of FUT2, encoding a fucosyltransferase counteracting with ST3GAL1/3/4 for sLex/a synthesis, was reduced. Knockdown and forced expression experiments showed that the induction of ST3GAL1/3/4 and FUT3 was mediated by c-Myc, most likely through its phosphorylation at Ser62 during EMT, and the suppression of FUT2 was attributed to the attenuation of CDX2 . These results suggest a pivotal involvement of c-Myc and CDX2 in sLex/a expression in colon cancer cells undergoing EMT. 2. CDX Transcription Factors Positively

Regulate Expression of PLEKHG1 in

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Intestinal Epithelium Aoki, M., Fujishita, T. and Taketo, MM. *1

The Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate development of the gut, as wells differentiation of intestinal epithelial cells. The CDX transcription factors are often down-regulated in colorectal carcinomas, and considered candidate tumor suppressors. A chromatin immunoprecipitation (ChIP) screen identified pleckstrin homology domain containing, family G (with RhoGef domain) member 1 (PLEKHG1) gene, a member of genes encoding Dbl-family RhoGEFs, as a novel target of CDX1 and CDX2. The CDX transcription factors bind to the upstream enhancer region of the PLEKHG1 gene and transactivate reporter genes containing the region. Overexpression of Cdx1 or Cdx2 in a normal rat intestinal epithelial cell line markedly induced expression of the endogenous Plekhg1, whereas knockdowns of CDX2 reduced the PLEKHG1 transcript level in human colon cancer cells. Lentivirus-mediated knockdown of PLEKHG1 in HCT 116 colon cancer cells resulted in enhanced proliferation and decreased cell-cell adhesion. Preliminary experiments using Plekhg1 knockout mice show that the loss of Plekhg1 enhances polyp formation in the colon of Apc +/Δ716 mice, a model for familial adenomatous polyposis (FAP) and the early stage of colon cancer development. These results suggest PLEKHG1 may have a tumor suppresser-like function in the colon.

*1 Department of Pharmacology, Graduate School of Medicine, Kyoto University 3. Roles of the mTOR signaling in

intestinal adenocarcinoma formation in cis-Apc/Smad4 mutant mice

Fujishita, T., Taketo, MM. *1 and Aoki, M.

The serine/threonine kinase mTOR (mammalian target of rapamycin) forms two distinct complexes: mTORC1 (mTOR complex 1) and mTORC2. Activation of mTORC1 has been implicated in various types of human cancers. We previously reported that the mTORC1-specific inhibitor everolimus (RAD001) suppressed the

formation of benign intestinal adenomas in Apc∆716

mice and prolonged their survival. However, we noticed that long-term treatment with RAD001 resulted in emergence of resistant tumors that showed hyperactivation of the mTORC2 pathway. To determine the roles of the mTORC pathways in formation of invasive intestinal adenocarcinomas, we treated cis-Apc∆716/Smad4 compound mutant mice (cis-Apc/Smad4 mice) with RAD001 (everolimus), an mTORC1 selective inhibitor, or AZD8055, an ATP-competitive kinase inhibitor of mTOR. RAD001 treatment suppressed intestinal adenocarcinoma formation and their local invasion, and improved their survival. While eight-week treatment was required to block adenocarcinoma formation with RAD001, two-week treatment with AZD8055 significantly suppressed it. AZD8055 treatment suppressed the mTORC2 activation in tumors of cis-Apc/Smad4 mice. Moreover, the phosphorylation level of 4E-BP1, a downstream target of mTORC1, in tumors was greatly reduced by AZD8055, as compared with RAD001. These results indicate the essential roles of the mTOR signaling in the growth of intestinal adenocarcinomas as well as in adenomas, and also potent antineoplastic activity of the mTOR kinase inhibitor against them.

*1 Department of Pharmacology, Graduate School of Medicine, Kyoto University

Fig.1. Schematic representation of the suppressive effects of the mTORC1 inhibitor RAD001 (A) , and the mTOR kinase inhibitor AZ8055 (B), on intestinal adenocarcinoma formation in cis-Apc/Smad4 mice.

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From left to right

First row: Ms. Y. Takada, Dr. S. Era, Ms. K. Kobori, Dr. H. Goto, Ms. N. Tanigawa Second row: Dr. H. Tanaka, Dr. H. Inaba, Dr. M. Inagaki, Ms. E. Kawamoto, Dr. K. Kasahara,

Dr. I. Izawa, Dr. Y. Hayashi, Dr. A. Inoko Inset: Dr. M. Matsuyama, Dr. D. He, Dr. Z. Wang, Ms. C. Yuhara, Ms. Y. Itoh, Dr. P. Li

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Division of Biochemistry ________________________________________________________________________________ Masaki Inagaki, M.D., Ph.D. Chief Ichiro Izawa, M.D., Ph.D. Section Head Hidemasa Goto, M.D., Ph.D. Section Head Akihito Inoko, M.D., Ph.D. Senior Researcher Kousuke Kasahara, Ph.D. Researcher Yuko Hayashi, Ph.D. Research Assistant Hiroki Tanaka, D.D.S., Ph.D. Research Resident Makoto Matsuyama, Ph.D. Research Resident (until March 2012) Dongwei He, Ph.D. Research Resident (until November 2012) Zhonghua Wang, Ph.D. Research Resident (until May 2012) Hironori Inaba, Ph.D. Research Resident (as of April 2013) Saho Era, Ph.D. Research Resident (as of April 2013) Kyoko Kobori, Semi-regular Employee Eriko Kawamoto, Semi-regular Employee Naomi Tanigawa, Semi-regular Employee (as of April 2013) Visiting Scientists Ping Li, M.D., Ph.D. Global COE Postdoctoral Fellow, Nagoya University Graduate School of Medicine General Summary

Aneuploidy, a phenotype of chromosomal instability, is a common genetic feature of solid human cancers, although it remains unclear whether as a cause or a consequence of malignant transformation. Recently, aneuploidy has been also reported to be involved in ageing. We generated knock-in mice featuring vimentin with mitotic phospho-defective mutations to impair cytokinesis. Homozygotic mice (VIMSA/SA) presented with microophthalmia and cataracts, in which lens epithelial cells exhibited chromosomal instability, including binucleation and aneuploidy, along with premature aging. VimentinSA/SA mice may offer beneficial tools to further dissect the molecular link between aneuploidy, tumorigenesis and aging.

Tumor cells are known to often lack primary cilia, but whether their loss is directly linked to tumorigenesis is completely unclear. We revealed that the trichoplein-Aurora A pathway is integral to continuous suppression of primary cilia assembly, which is required for G1 cell cycle progression. In addition, we have recently found that the ubiquitin proteasome pathway is involved in the regulation of primary cilia formation.

In addition, to investigate the molecular mechanisms of cell cycle progression and chromosomal instability, we have been examining the functions of kinases including Chk1, Plk1, Aurora A, and Aurora B, taking advantage of the availability of site- and phosphorylation state-specific antibodies. 1. Emerging roles of ubiquitin-proteasome machinery in formation of primary cilia Kasahara, K., Era, S., Kawamoto, E., Kawakami, Y.*1, Kiyono, T.*2, Kawamura, Y.*3, Goshima, N.*1, and Inagaki, M.

Non-motile primary cilia are microtubule-based sensory organelles that regulate a number of signaling pathways during development and tissue homeostasis. Defects in primary cilia formation (ciliogenesis) result in numerous diseases and disorders commonly known as ciliopathies. Primary cilia are grown from modified centrioles called basal bodies in response to cell cycle exit or entry into quiescence, and this process is achieved

through coordinated functions between positive and negative regulators of ciliogenesis. However, the regulatory mechanisms of ciliogenesis remain to be disclosed in detail.

The ubiquitin-proteasome system (UPS) controls many aspects of cell physiology including cell cycling and organelle biogenesis. In this study, we newly recognized a role of UPS in primary cilia biogenesis. Using human cells, we could show that proteasome activity is essential for ciliogenesis and destruction of Trichoplein, a centriolar protein that negatively regulates ciliogenesis through activation of Aurora-A kinases. Trichoplein is rapidly degraded in a polyubiquitylation-dependent fashion

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upon serum starvation. Using protein array and siRNA screens, we identified KCTD17, a protein that is required for ciliogenesis, as a ubiquitin E3 ligase for Trichoplein from 1172 putative E3 ligases. Mechanistically, KCTD17 functions as a substrate-adaptor of Cul3-based ubiquitin E3 ligase that polyubiquitylates Trichoplein. SiRNA mediated KCTD17 depletion prevented Trichoplein degradation and inactivation of Aurora-A, thereby leading to defective ciliogenesis after serum starvation. These phenotypes were rescued by expression of siRNA-resistant Myc-tagged KCTD17 or co-silencing of Trichoplein, and recapitulated by expression of non-degradable Trichoplein mutant. Similar to KCTD17 loss, Cul3 depletion also blocked ciliogenesis and Trichoplein degradation. These results indicate that Cul3-KCTD17 ubiquitin E3 ligase promotes ciliogenesis through destruction of Trichoplein.

Maintenance of normal Trichoplein levels is essential for cell cycle progression through regulating ciliogenesis, as excessive Trichoplein suppresses aberrant ciliogenesis in non-proliferating cells, whereas its depletion leads to aberrant ciliogenesis in proliferating cells. Our study reveals that UPS controls degradation of Trichoplein to promotes ciliogenesis. We propose that UPS deficiency serves as a potential underlying mechanism of ciliogenesis, and that UPS regulation might provide a novel means of ciliopathy treatment. *1 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology *2 Virology Division, National Cancer Center Research Institute *3 Japan Biological Informatics Consortium (JBiC) 2. Cytokinetic failure induces aneuploidy

and aging in vimentin phosphorylation deficient mice

Tanaka, Hir., Matsuyama, M., Inoko, A., Goto, H., Yonemura, S. *1, Kobori, K., Tanigawa, N., Hayashi, Y., Kondo, E. *2, Itohara, S. *3, Izawa, I. and Inagaki, M.

Intermediate filaments (IFs), together with microtubules and actin filaments, form the cytoskeletal framework in the cytoplasm of eukaryotic cells. Unlike those of the two other major cytoskeletal networks, IF component proteins are divided into six groups whose composition generally depends on the tissue type and differentiation step. On the other hand, vimentin, a type III IF protein conserved in vertebrate evolution, is expressed ubiquitously in mesenchymal cells, with especially high levels in lens tissue.

There is increasing evidence that IF disassembly is regulated by phosphorylation of Ser/Thr residues in the amino-terminal head domains of IF proteins. Site- and phosphorylation state-specific antibodies that can recognize a phosphorylated residue and its flanking sequence are powerful tools to demonstrate site (domain)-specific IF phosphorylation in cells. Using such antibodies, we first showed that mitotic IF phosphorylation is regulated by distinct protein kinases in a spatiotemporally regulated manner. Further detailed studies revealed that several mitotic kinases such as Aurora-B, Cdk1, Plk1, and Rho-kinase, participate in mitotic phosphorylation of type III IF proteins. By transient expression of type III IF proteins mutated at these mitotic phosphorylation sites to Ala in a type III IF-deficient T24 cell line, we found that disturbance of mitotic IF phosphorylation induced abnormal IF structures (referred as IF bridges) connecting two daughter cells during the cytokinetic process. These findings indicated that mitotic IF phosphorylation is essential for efficient separation of IFs into two daughter cells. Cells with IF bridges appeared to make two distinct decisions regarding cell fate. IF bridges were torn off between two daughter cells, likely by cell-adhesion-dependent traction forces, completion of cytokinesis resulted. The other type of cells failed in cytokinesis, with formation of binucleate (multinucleate) cells. However, the significance of mitotic IF phosphorylation during organogenesis and tissue homeostasis remains largely unknown.

In the present study, we generated knock-in mice expressing vimentin that harbor mutations in mitotic phosphorylation sites. Homozygotic mice (VIMSA/SA) presented with microophthalmia and lens cataracts, whereas heterozygotic mice (VIMWT/SA) were indistinguishable from WT (VIMWT/WT) mice. In VIMSA/SA mice, lens epithelial cells were reduced in number and exhibited chromosomal instability (CIN), including binucleation and aneuploidy. Electron microscopic analyses revealed that lens fiber cells of VIMSA/SA mice exhibited membrane disorganization similar to defects in age-related cataracts. Since the lens mRNA level of a senescence (aging)-related gene was significantly elevated in VIMSA/SA, the lens phenotypes in VIMSA/SA suggested a possible causal relationship between CIN and premature aging. *1 Electron Microscope Laboratory, RIKEN Center for Developmental Biology *2 Division of Oncological Pathology, Aichi Cancer Center Research Institute

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*3 Laboratory for Behavioral Genetics, RIKEN Brain Science Institute 3. Perspective of Aurora A kinase as a

therapeutic target for cancer Goto, H., Watanabe, N.*1, Kobori, K., Inoko, A., Mochizuki, H.*2, Togashi, T.*2, Kisu, Y.*3, Kawamura, Y.*2, Kawakami, Y.*3, Goshima, N.*3, and Inagaki, M.

Aurora A (AURKA), a centrosomal mitotic kinase, controls many aspects of mitosis, such as entry into mitosis, centrosome maturation/separation, and bipolar spindle formation. Genetic amplification and protein overexpression of Aurora A are observed in many types of solid tumors and such up-regulation is associated with chromosomal instability (CIN). Aurora A kockdown (KD) or inhibition induces catastrophic CIN in cancerous cells, which results in mitotic cell death (mitotic catastrophe). More recently, we found that phenotypes of Aurora A KD are different between normal dupuloid and cancerous cells. Aurora A KD induces primary cilia formation in RPE1 (normal dupuloid) cells, while also arresting the cell cycle at the G0/G1-S transition in a ciliation-dependent manner. On the other hand, Aurora A KD failed to induce ciliogenesis or cell cycle arrest in HeLa cells, which have a reduced tendency to form primary cilia, like the majority of cancer cells. Based on these properties, we consider that Aurora A is a high-value target for cancer therapeutics.

Now, we are employing the following 2 strategies to inhibit Auora A-mediated signaling pathways. One is the screening for novel Aurora A inhibitors. Since Aurora A is activated through the binding of Aurora A-associated proteins such as TPX2 or trichoplein (TCHP), we are now searching for small chemical(s) to inhibit their interaction. We have already established a high-throughput screening system. The other is the screening for novel Aurora A-interacting proteins at centrosomes in order to identify more attactive molecular targets downstream of Aurora A. We have already obtained evidence for several putative Aurora A binding proteins, using protein arrays. We believe that these approaches will help to establish novel molecular targeted therapies. *1 RIKEN, Bioprobe Application Research Unit, Japan *2 Japan Biological Informatics Consortium (JBiC), Japan

*3 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Japan 4. Novel cell cycle regulation through

centrosomes Inoko, A., Inaba, H., He, D., Goto, H., Hayashi, Y., Izawa, I., Urano, T.*1, Yonemura, S.*2, Kiyono, T.*3, Goshima, N.*4, and Inagaki, M.

The cell cycle is characterized by a complex set of processes which lead to cell division accompanied by DNA duplication. The behavior of the centrosomes, and particularly the centrioles, is well synchronized with the cell cycle, but the mechanisms are not fully understood. Recently, we obtained clues from kinetics of primary cilia, that protrude from mother centrioles in cultured cells, especially in cellular quiescence.

We found that trichoplein, previously reported as keratin-binding protein, also localizes to the centrioles in proliferating cells. Interestingly, serum starvation causes ciliated mother centrioles to lose trichoplein. Ciliation was inhibited by exogenous expression of trichoplein. In addition, trichoplein depletion was found to accelerate primary cilia assembly accompanied with G1 arrest even with serum cultivation. Notably, this G1 arrest was reversed by a treatment for abrogating cilia. Finally, it was established that the underlying mechanism is centriolar Aurora A (AurA) activation by trichoplein in G1 phase. Thus, the trichoplein-AurA pathway in the centrosome acts as a switch for cell cycle progression through regulation of primary cilia kinetics.

Based on this finding, we searched for similar proteins to trichoplein in combination with amino acid motif, determined localization, and estimated functions by siRNA screening. Currently, we have several candidate genes that mimic the phenotype of trichoplein depletion. Interestingly, some of them accomplished G1 arrest without primary cilia assembly. In this regard, it is of interest that other groups previously reported that loss of centrosome integrity induces p38–p53–p21 dependent G1-S arrest, but our present phenotype is combined with intact centrosomes, suggesting the involvement of novel mechanisms. Investigation of the underlying mechanisms are ongoing. *1 Department of Biochemistry, Shimane University School of Medicine *2 RIKEN Center for Developmental Biology *3 Virology Division, National Cancer Center Research Institute *4 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Japan 5. Interaction of Cell Polarity Regulator

Scribble with Multidrug Resistance Protein 4 (MRP4/ABCC4)

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Izawa, I., Hayashi, Y., and Inagaki, M. Scribble, originally identified as a tumor

suppressor in Drosophila, has been implicated in the regulation of many aspects of cellular physiology, including apical-basal cell polarity, migration, proliferation, and vesicle trafficking. To further elucidate the functions of human Scribble, we performed a yeast two-hybrid screen to search for binding partners and identified Multidrug Resistance Protein 4 (MRP4/ABCC4) as one potential example. MRP4 is a transmembrane protein which is involved in substrate-specific transport of endogenous and exogenous substrates, including anti-cancer drugs. In vitro binding assays

revealed direct binding to the first PDZ domain (PDZ1) of Scribble through its C-terminus. By coimmunoprecipitation assays, we confirmed interaction of endogenous MRP4 and Scribble. Immunofluorescence further revealed Scribble co-localization with MRP4 at basolateral membranes. In Caco-2 and LNCaP cells, depletion of Scribble by siRNAi impaired the plasma membrane localization of MRP4. These results suggest that Scribble may affect drug resistance through regulation of basolateral membrane targeting of MRP4.

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From left to right

First row: Mr. Isogai S, Dr. Tojo M, Dr. Shinjo K, Dr. Kondo Y, Ms. Kitao M, Ms. Sasaki A, Ms. Takagi N

Second row: Dr. Katsushima K, Dr. Ichimura N, Dr. Ohka F, Dr. Hatanaka A, Mr. Oiwa Y, Dr. Tu M

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Division of Epigenomics ________________________________________________________________________________ Yutaka Kondo M.D., Ph.D., Chief Keiko Shinjo, M.D., Ph.D., Senior Researcher Michiko Kitao, Semi-regular Employee Nami Takagi, Semi-regular Employee Akiko Sasaki, Semi-regular Employee Yuki Oiwa, Semi-regular Employee Research Resident Humiharu Ohka, M.D., Ph.D. Keisuke Katsushima, Ph.D. Akira Hatanaka, Ph.D. Visiting Trainees Norihisa Ichimura, D.D.S., Nagoya University School of Medicine Masayuki Tojo, M.D., Showa University School of Medicine Shinya Sato, M.D., Ph.D., Nagoya City University School of Medicine Kota Ishikawa, M.D., Nagoya University School of Medicine Shuntaro Isogai, Nagoya City University School of Medicine Min Tu, M.D., Ph.D., Nanjing Medical University Yasuyuki Okamoto, M.D., Ph.D., Nagoya City University School of Medicine (until September 2013) General Summary

Alongside known genetic changes, aberrant epigenetic alteration has emerged as common hallmark of many cancers. Epigenetic mechanisms are important in tumor progression but also possibly in tumor formation. Early detection of cancer-specific epigenetic alterations as well as therapies targeted at specific epigenetic regulators or their effectors may improve patient outcomes. Since the clinical impact of biomarkers is dependent on the stability of the information provided, DNA methylation is an ideal biomarker for cancer diagnosis. We explored the epigenetic landscapes in many types of cancers and found that DNA methylation of certain genes predominantly reflects the characteristic clinicopathological features of tumors. In order to apply these markers for clinical use, we have invented a novel and highly sensitive technology to detect methylated DNA in samples obtained through less invasive procedures, such as serum.

Cancers are mostly comprised of heterogeneous cell populations. A significant degree of morphological and lineage heterogeneity may contribute to tumor invasion, metastasis and drug resistance. Since the epigenome may act at the top of the hierarchy of gene control mechanisms, dysregulation of epigenetics essentially has effects on multiple pathways relevant to the cancer phenotype. Cancer stem cells (CSC) may usurp a plastic epigenetic mechanism as a mediator of adaptation to their environment, which can contribute to tumor heterogeneity. Our works is focused on dynamic epigenetic mechanisms in cancers, their relationship with aberrant gene expression, and their potential for applications in drug discovery. In particular, we are targeting epigenetic plasticity, a challenging potentially effective approach for cancer treatment. 1. The PRC2 chromatin regulator is a key

regulator of epigenetic plasticity in glioblastoma

Natsume, A.*1, Ito, M.*1, Katsushima, K., Ohka, F., Hatanaka, A., Shinjo, K.*2, Sato, S.*3, Takahashi, S.*3, Ishikawa, Y.*4, Takeuchi, I.*4, Shimogawa, H.*5, Uesugi, M.*5, Okano, H.*6, Kim, SU.*7, Wakabayashi, T.*1, Issa, JP.*8, Sekido, Y.*9, and Kondo, Y.

Tumor cell plasticity contributes to functional and morphologic heterogeneity. To uncover the

underlying mechanisms, we examined glioma stem-like cells (GSC) where we found that biologic interconversion between GSCs and differentiated non-GSCs is functionally plastic and accompanied by gain or loss of polycomb repressive complex 2 (PRC2), a complex that modifies chromatin structure. PRC2 mediates lysine 27 trimethylation on histone H3 and in GSCs it affects pluripotency or development-associated genes (e.g., Nanog,

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Wnt1, and BMP5) together with alterations in the subcellular localization of EZH2, a catalytic component of PRC2. Intriguingly, exogenous expression of EZH2-dNLS, which lacks nuclear localization sequence, impaired the repression of Nanog expression under differentiation conditions. RNA interference (RNAi)-mediated attenuation or pharmacologic inhibition of EZH2 had little to no effect on apoptosis or bromodeoxyuridine incorporation in GSCs, but it disrupted morphologic interconversion and impaired GSC integration into brain tissue, thereby improving survival of GSC-bearing mice. Pathologic analysis of human glioma specimens revealed that the number of tumor cells with nuclear EZH2 is larger around tumor vessels and at invasive fronts, suggesting that nuclear EZH2 may help reprogram tumor cells in close proximity to particular microenvironments. Our results indicate that epigenetic regulation by PRC2 is a key mediator of tumor cell plasticity, which is required for the adaptation of glioblastoma cells to their microenvironment. Thus, PRC2-targeted therapy may reduce tumor cell plasticity and tumor heterogeneity, offering a new paradigm for glioma treatment. *1Department of Neurosurgery, Nagoya University School of Medicine *2Division of Oncological Pathology, Aichi Cancer Center Research Institute *3Department of Experimental Pathology and Tumor

Biology, Nagoya City University Graduate School of Medical Sciences *4Department of Scientific and Engineering Simulation, Graduate School of Engineering, Nagoya Institute of Technology *5Institute for Integrated Cell-Material Sciences/ Institute for Chemical Research, Kyoto University *6Department of Physiology, Keio University School of Medicine *7Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, Canada; Medical Research Institute, Chungang University College of Medicine *8Fels Institute for Cancer and Molecular Biology Temple University *9Division of Molecular Oncology, Aichi Cancer Center Research Institute 2. Hepatitis Virus Infection Affects DNA

Methylation in Mice with Humanized Livers.

Okamoto Y, Shinjo K*1, Shimizu Y*2, Sano T*2, Yamao K*3, Gao W*4, Fujii M*5, Osada H*5, Sekido Y*5, Murakami S*6, Tanaka Y*6, Joh T*7, Sato S*8, Takahashi S*8, Wakita T*9, Zhu J*10, Issa JP*11, and Kondo Y.

Fig. 1. Targets of PRC2-mediated H3K27me3 during phenotypic conversion of GSCs and S-BTCs. A, Convertible Nestin promoter activity in 1228-GSCs-pE-Nes. GSC lines stably expressing the GFP under the Nestin promoter using a pE/nestin:EGFP construct (GSC-pE-Nes) were generated. 1228-GSCs-pE-Nes lost EGFP expression by 96 hours in serum-containing culture, and re-expressed EGFP following return to NBE for 72 hours. Bar, 100 μm. B, H3K27me3 enrichment at BMP5 and Wnt1 promoters in GSCs and S-BTCs. The y axis represents enrichment of H3K27me3 (relative value of H3K27me3 to H3). C, Brain-slice assays. 1228-GSCs transduced with either mock control (Ctrl), EZH2-shRNA (EZH2 KD), Wnt1-shRNA (Wnt1 KD) or BMP5-overexpression (BMP5-expression) were placed in the basal ganglia of nude mice for 14 days (top panel). From immunofluorescence images (middle panel), expression indices of GFAP and Nestin were calculated (bottom panel). The Y axis indicates values of (Intensity-background, mean ± SD) x ROI. D, 1228-GSCs-pE-Nes were cultured for 7 days on mouse brain slices. Dashed line indicates border of sphere. Arrowheads indicate differentiated tumor cells spread into the surrounding brain tissue. The areas (a) and (b) in the merged image (bottom left) are magnified in panels (a) and (b).

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Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas (HCCs). Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells following hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (uPA/SCID mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV; liver tissues were collected and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of HCC and adjacent non-tumor liver tissues from patients. No reproducible changes in DNA methylation were observed following infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected uPA/SCID

mice. There were changes in 160±63 genes in HBV-infected and 237±110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in HCC samples from patients, compared with non-tumor tissues. Expression of Ifng, which is expressed by natural killer (NK) cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced following administration of an inhibitor of NK cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appear to activate an NK cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. *1Division of Oncological Pathology, Aichi Cancer Center Research Institute *2Department of Gastroenterological Surger, Aichi Cancer Center Hospital *3Department of Gastroenterology, Aichi Cancer Center Hospital *4Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University *5Division of Molecular Oncology, Aichi Cancer Center Research Institute *6Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences *7Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences *8Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences *9Department of Virology II, National Institute of Infectious Diseases

Fig. 2. Hepatitis Virus Infection Affects DNA Methylation in Mice with Humanized Livers. Recent technological advances have enabled development of severe combined immunodeficiency (SCID) mice carrying a urokinase-type plasminogen activator (uPA) transgene controlled by an albumin promoter (uPA/SCID), in which the liver is repopulated with human hepatocytes (human hepatocyte chimeric mice). This mouse model features severe combined immunodeficiency due to lack of both T- and B-cell, but with normal macrophages and natural killer cell (NK cell) activity, which are important components of the innate immune system. Since both HBV and HCV can infect human hepatocytes but not murine hepatocytes, this model is a useful tool for mimicking and unraveling hepatitis virus-host interactions in vivo. Using this model, we demonstrated here that DNA methylation was induced in human hepatocytes after HBV and HCV infection, and that induction of DNA methylation was closely associated with NK cell activity.

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*10Cancer Epigenetics Program, Shanghai Cancer Institute, Shanghai Jiaotong University *11Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine

AACR-JCA meeting 2013

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From left to right Dr. M. Inagaki, Dr. H. Kumimoto, Mr. Y. Minoura, Mr. Y. Nishikawa, Ms. Y. Shinohara, and Dr. H. Nakamura

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Central Service Unit ________________________________________________________________________________ Masaki Inagaki, M.D. Chief Hiroshi Kumimoto, Ph.D. Senior Researcher Yasushi Minoura, B.P., Senior Research Assistant Harunari Tanaka, B.P. Research Assistant (until March 2012) Hiromu Nakamura, D.M.Sc. Senior Semi-regular Research Assistant Yasuhiro Nishikawa, Semi-regular Research Assistant Yoshimi Shinohara, Semi-regular Employee (as of August 2013) Visiting Trainees Erika Ito, M.D. School of Medicine, Nagoya City University General Summary Our main research project is molecular epidemiologic analysis of human esophageal cancer. Especially, we have focused on the relationship between numbers of polymorphisms in the D-loop region of mitochondrial DNA (mtDNA) and risk of esophageal cancer development. 1. Relationship between risk of

esophageal cancer and the number of polymorphisms in mitochondrial DNA

Kumimoto, H. Mitochondria are well known as the organelles

in human for production of energy for cells and also they have roles in apoptosis. Recently, frequent mutations have been found in various types of cancer, including examples in the breast and stomach. Our previous analysis of mutations in the D-loop region of mitochondrial DNA (mtDNA) in esophageal tumors demonstrated frequent somatic mutations (in 34 % of cases). We also determined nuclear genomic instability, but did not find any correlation with somatic mtDNA mutations, suggesting that instability of mtDNA in esophageal cancer might be an independent of nuclear genomic instability.

Energy as ATP is produced in mitochondria with reactive oxygen species (ROS) as a byproduct. Polymorphisms in the genes related to oxidative phosphorylation may elevate ROS production by leaking electrons. Therefore the number of polymorphisms in mtDNA may influence ROS

levels in cells, which would be expected to increase the risk of introducing mutations into mtDNA and genomic DNA. We therefore analyzed the number of polymorphisms in mtDNA as a surrogate marker for ROS, then evaluating the relationship with risk of esophageal cancer.

We performed sequencing analysis of D-loop region in mtDNA using DNA samples from esophageal cancer subjects and non-cancer controls collected in the HERPACC study. At first, we used re-sequencing primers sets made by ABI, mitoSEQr. We found that the whole D-loop region could be sequenced with 4 of 8 primer sets. After sequencing the whole D-loop region, we identified polymorphisms by comparing these sequences with the common mtDNA sequence.

So far, we have completed analyses of polymorphisms in 33 subjects with esophageal cancer and 64 non-cancer controls (see Table 1). After analyses of polymorphisms in 185 esophageal cancer patients and 185 non-cancer controls, we will evaluate the relationship between esophageal cancer risk and number of mitochondrial polymorphisms.

Table 1. Summary of analyses of polymorphism in mtDNA

Data are shown as average numbers / subject except 'number of subjects analyzed'.

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Librarians ________________________________________________________________________________

From left to right

Ms. T. Yasuda, Ms. T. Shibata, Mr. T. Matsunaga, Ms. M. Sakou, Ms. N. Terashima

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Publications ________________________________________________________________________________ Journals

J001. Abe, S., Morita, Y., Kaneko, M.K., Hanibuchi, M., Tsujimoto, Y., Goto, H., Kakiuchi, S., Aono, Y., Huang, J., Sato, S., Kishuku, M., Taniguchi, Y., Azuma, M., Kawazoe, K., Sekido, Y., Yano, S., Akiyama, S., Sone, S., Minakuchi, K., Kato, Y., Nishioka, Y.: A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody. J Immunol, 190: 6239-6249, 2013. (PMID: 23690472)

J002. Akatsuka, S., Yamashita. Y., Ohara, H., Liu, Y., Izumiya, M., Abe, K., Ochiai, M., Jiang, L., Nagai, H., Okazaki, Y., Murakami, H., Sekido, Y., Arai, E., Kanai, Y., Hino, O., Takahashi, T., Nakagama, H., Toyokuni, S.: Fenton reaction induced cancer in wild type rats recapitulates genomic alterations observed in human cancer. PLoS One, 7: e43403, 2012. (PMID: 22952676)

J003. Akita, K., Yoshida, S., Ikehara, Y., Shirakawa, S., Toda, M., Inoue, M., Kitawaki, J., Nakanishi, H., Narimatsu, H., Nakada, H.: Different levels of sialyl-Tn antigen expressed on MUC16 in patients with endometriosis and ovarian cancer. Int J Gynecol Cancer, 22: 531-538, 2012 (PMID: 22367369)

J004. Arita, K., Maeda-Kasugai, Y., Ohshima, K., Tsuzuki, S., Suguro-Katayama, M., Karube, K., Yoshida, N., Sugiyama, T., Seto, M.: Generation of mouse models of lymphoid neoplasm using retroviral gene transduction of in vitro-induced germinal center B and T cells. Exp Hematol, 41: 731-41.e9, 2013. (PMID: 23583576)

J005. Asai, H., Fujiwara, H., An, J., Ochi, T., Miyazaki, Y., Nagai, K., Okamoto, S., Mineno, J., Kuzushima, K., Shiku, H., Inoue, H., Yasukawa, M.: Co-introduced functional CCR2 potentiates in vivo anti-lung cancer functionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor. PLoS One, 8: e56820, 2013. (PMID: 23441216)

J006. Bojesen, S., Pooley, K., Johnatty, S., Beesley, J., Michailidou, K., Tyrer, J., Edwards, S., Pickett, H., Shen, H., Smart, C., Hillman, K., Mai, P., Lawrenson, K., Stutz, M., Lu, Y., Karevan, R., Woods, N., Johnston, R., French, J., Chen, X., Wesicher, M., Nielsen, S., Maranian, M., Ghoussaini, M., Ahmed, S., Baynes, C., Humphreys, M., Wang, J., Dennis, J., McGuffog,

L., Barrowdale, D., Lee, A., Healey, S., Lush, M., Tessier, D., Vincent, D., Bacot, F., Australian Cancer Study, Australian Ovarian Cancer Study Group, Vergote, I., Lambrechts, S., Despierre, E., Risch, H., Gonzlez-Neira, A., Rossing, M., Pita, G., Doherty, J., Dvarez, N., Larson, M., Fridley, B., Schoof, N., Chang-Claude, J., Cicek, M., Peto, J., Kalli, K., Broeks, A., Armasu, S., Schmidt, M., Braaf, L., Winterhoff, B., Nevanlinna, H., Konecny, G., Lambrechts, D., Rogmann, L., Gunel, P., Teoman, A., Milne, R., Garcia, J., Cox, A., Shridhar, V., Burwinkel, B., Marme, F., Hein, R., Sawyer, E., Haiman, C., Wang-Gohrke, S., Andrulis, I., Moysich, K., Hopper, J., Odunsi, K., Lindblom, A., Giles, G., Brenner, H., Simard, J., Lurie, G., Fasching, P., Carney, M., Radice, P., Wilkens, L., Swerdlow, A., Goodman, M., Brauch, H., Garca-Closas, M., Hillemanns, P., Winqvist, R., Drst, M., Devilee, P., Runnebaum, I., Jakubowska, A., Lubinski, J., Mannermaa, A., Butzow, R., Bogdanova, N., Drk, T., Pelttari, L., Zheng, W., Leminen, A., Anton-Culver, H., Bunker, C., Kristensen, V., Ness, R., Muir, K., Edwards, R., Meindl, A., Heitz, F., Matsuo, K., Bois, A., Wu, A., Harter, P., Teo, S., Schwaab, I., Shu, X., Blot, W., Hosono, S., Kang, D., Nakanishi, T., Hartman, M., Yatabe, Y., Hamann, U., Karlan, B., Sangrajrang, S., Kjaer, S., Gaborieau, V., Jensen, A., Eccles, D., Hgdall, E., Shen, C., Brown, J., Woo, Y., Shah, M., Mat, Adenan, N., Luben, R., Omar, S., Czene, K., Vierkant, R., Nordestgaard, B., Flyger, H., Vachon, C., Olson, J., Wang, X., Levine, D., Rudolph, A., Weber, R., Flesch-Janys, D., Iversen, E., Nickels, S., Schildkraut, J., Dos-Santos-Silva, I., Cramer, D., Gibson, L., Terry, K., Fletcher, O., Vitonis, A., van der Schoot, E., Poole, E., Hogervorst, F., Tworoger, S., Liu, J., Bandera, E., Li, J., Olson, S., Humphreys, K., Orlow, I., Blomqvist, C., Rodriguez-Rodriguez, L., Aittomki, K., Salvesen, H., Muranen, T., Wik, E., Brouwers, B., Krakstad, C., Wauters, E., Halle, M., Wildiers, H., Kiemeney, L., Mulot, C., Aben, K., Laurent-Puig, P., van Altena, A., Truong, T., Massuger, L., Benitez, J., Pejovic, T., Arias, Perez, J., Hoatlin, M., Zamora, M., Cook, L., Balasubramanian, S., Kelemen, L., Schneeweiss, A., Le, N., Sohn, C., Brooks-Wilson, A., Tomlinson, I., Kerin, M., Miller, N., Cybulski, C., kConFab Investigators, Henderson, B., Menkiszak, J., Schumacher, F., Wentzensen, N., Le Marchand, L., Yang, H., Mulligan, A., Glendon, G., Engelholm, S., Knight, J., Hgdall, C., Apicella, C., Gore, M., Tsimiklis, H., Song, H., Southey, M.,

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Jager, A., van den Ouweland, A., Martens, J., Flanagan, J., Kriege, M., Paul, J., Margolin, S., Siddiqui, N., Severi, G., Whittemore, A., Baglietto, L., McGuire, V., Stegmaier, C., Sieh, W., Mller, H., Arndt, V., LabrChe, F., Gao, Y., Goldberg, M., Yang, G., Dumont, M., McLaughlin, J., Hartmann, A., Ekici, A., Beckmann, M., Phelan, C., Lux, M., Permuth-Wey, J., Peissel, B., Sellers, T., Ficarazzi, F., Barile, M., Ziogas, A., Ashworth, A., Gentry-Maharaj, A., Jones, M., Ramus, S., Orr, N., Menon, U., The Genica Network, Pearce, C., Brning, T., Pike, M., Ko, Y., Lissowska, J., Figueroa, J., Kupryjanczyk, J., Chanock, S., Dansonka-Mieszkowska, A., Jukkola-Vuorinen, A., Rzepecka, I., Pylks, K., Bidzinski, M., Kauppila, S., Hollestelle, A., Seynaeve, C., Monteiro, A., Tollenaar, R., Durda, K., Jaworska, K., Hartikainen, J., Kosma, V., Kataja, V., Antonenkova, N., Long, J., Shrubsole, M., Deming-Halverson, S., Lophatananon, A., Siriwanarangsan, P., Stewart-Brown, S., Ditsch, N., Lichtner, P., Schmutzler, R., Ito, H., Iwata, H., Tajima, K., Tseng, C., Stram, D., den, D., Yip, C., Ikram, M., The, Y., Cai, H., Lu, W., Signorello, L., Cai, Q., Noh, D., Yoo, K., Miao, H., Iau, P., Teo, Y., McKay, J., Shapiro, C., Ademuyiwa, F., Fountzilas, G., Hsiung, C., Yu, J., Hou, M., Healey, C., Luccarini, C., Wang, Q., Peock, S., Stoppa-Lyonnet, D., Peterlongo, P., SWE-BRCA, Rebbeck, T., Piedmonte, M., Singer, C., Friedman, E., Thomassen, M., Offit, K., Hansen, T., Neuhausen, S., Szabo, C., Blanco, I., Garber, J., Narod, S., Weitzel, J., Montagna, M., Olah, E., Godwin, A., Yannoukakos, D., Goldgar, D., Caldes, T., Imyanitov, E., Tihomirova, L., Arun, B., Campbell, I., Mensenkamp, A., van Asperen, C., van Roozendaal, K., Meijers-Heijboer, H., HEBON, Colle, J., Oosterwijk, J., Hooning, M., Rookus, M., van der Luijt, R., van Os, T., Evans, D., Frost, D., Fineberg, E., Embrace, Barwell, J., Walker, L., Kennedy, M., Platte, R., Davidson, R., Ellis, S., Cole, T., de Paillerets, B., Buecher, B., Damiola, F., Collaborators, G., Faivre, L., Frenay, M., Sinilnikova, O., Caron, O., Giraud, S., Mazoyer, S., Bonadona, V., Caux-Moncoutier, V., Toloczko-Grabarek, A., Gronwald, J., Byrski, T., Spurdle, A., Bonanni, B., Zaffaroni, D., Giannini, G., Bernard, L., Dolcetti, R., Manoukian, S., Norbert, A., Engel, C., Helmut, D., Rhiem, K., Alfons, M., Dieter, N., Hansjoerg, P., Christian, S., Wappenschmidt, B., Borg, A., Melin, B., Rantala, J., Soller, M., Nathanson, K., Domchek, S., Rodriguez, G., Salani, R., Kaulich, D., Tea, M., Paluch, S., Laitman, Y., Skytte, A., Kruse, T., Jensen, U., Robson, M., Gerdes, A., Ejlertsen, B.,

Foretova, L., Savage, S., Lester, J., Soucy, P., Kuchenbaecker, K., Olswold, C., Cunningham, J., Slager, S., Pankratz, V., Dicks, E., Lakhani, S., Couch, F., Hall, P., Gayther, S., Pharoah, P., Reddel, R., Goode, E., Greene, M., Easton, D., Berchuck, A., Antoniou, A., Chenevix-Trench, G., Dunning, A.: Multiple independent TERT variants associated with telomere length and risks of breast and ovarian cancer. Nat Genet, 45: 371-384, 2013. (PMID: 23535731)

J007. Chew, S.H., Okazaki, Y., Nagai, H., Misawa, N., Akatsuka, S., Yamashita, K., Jiang, L., Yamashita, Y., Noguchi, M., Hosoda, K., Sekido, Y., Takahashi, T., Toyokuni, S.: Cancer-promoting role of adipocytes in asbestos-induced mesothelial carcinogenesis through dysregulated adipocytokine production. Carcinogenesis, 2013 Sep 10, [Epub ahead of print] (PMID: 23917077)

J008. Chihara, D., Ito, H., Katanoda, K., Shibata, A., Matsuda, T., Tajima, K., Sobue, T., Matsuo, K.: Increase in incidence of adult T-cell leukemia/lymphoma in non-endemic areas of Japan and the United States. Cancer Sci, 103: 1857-1860, 2012. (PMID: 22738276)

J009. Chihara, D., Ito, H., Matsuda, T., Katanoda, K., Shibata, A., Saika, K., Sobue, T., Matsuo, K.: Decreasing trend in mortality of chronic myelogenous leukemia patients after introduction of imatinib in Japan and the U.S. Oncologist, 17: 1547-1550, 2012. (PMID: 22971523)

J010. Chihara, D., Matsuo, K., Kanda, J., Hosono, S., Ito, H., Nakamura, S., Seto, M., Morishima, Y., Tajima, K., Tanaka, H.: Inverse association between soy intake and non-Hodgkin lymphoma risk among women: a case-control study in Japan. Ann Oncol, 23: 1061-1066, 2012. (PMID: 21765045)

J011. Chuang, S.C., Jenab, M., Heck, J.E., Bosetti, C., Talamini, R., Matsuo, K., Castellsague, X., Franceschi, S., Herrero, R., Winn, D.M., La, Vecchia, C., Morgenstern, H., Zhang, Z.F., Levi, F., Dal, Maso, L., Kelsey, K., McClean, M.D., Vaughan, T., Lazarus, P., Muscat, J., Ramroth, H., Chen, C., Schwartz, S.M., Eluf-Neto, J., Hayes, R.B., Purdue, M., Boccia, S., Cadoni, G., Zaridze, D., Koifman, S., Curado, M.P., Ahrens, W., Benhamou, S., Matos, E., Lagiou, P., Szeszenia-Dabrowska, N., Olshan, A.F., Fernandez, L., Menezes, A., Agudo, A., Daudt, A.W., Merletti, F., Macfarlane, G.J., Kjaerheim, K., Mates, D., Holcatova, I., Schantz, S., Yu, G.P., Simonato, L., Brenner, H., Mueller, H., Conway,

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D.I., Thomson, P., Fabianova, E., Znaor, A., Rudnai, P., Healy, C.M., Ferro, G., Brennan, P., Boffetta, P., Hashibe, M.: Diet and the risk of head and neck cancer: a pooled analysis in the INHANCE consortium. Cancer Cause Control, 23: 69-88, 2012 (PMID: 22037906)

J012. De, Feo, E., Simone, B., Kamgaing, R.S., Gallì, P., Hamajima, N., Hu, Z., Li, G., Li, Y., Matsuo, K., Park, J.Y., Roychoudhury, S., Spitz, M.R., Wei, Q., Zhang, J.H., Ricciardi, W., Boccia, S.: p73 G4C14-to-A4T14 gene polymorphism and interaction with p53 exon 4 Arg72Pro on cancer susceptibility: a meta-analysis of the literature. Mutagenesis, 27: 267-273, 2012. (PMID: 21976716)

J013. Demachi-Okamura, A., Torikai, H., Akatsuka, Y., Miyoshi, H., Yoshimori, T., Kuzushima, K.: Autophagy creates a CTL epitope that mimics tumor-associated antigens. PLoS One, 7: e47126, 2012. (PMID: 23071732)

J014. Duverle, D.A., Takeuchi, I., Murakami-Tonami, Y., Kadomatsu, K., Tsuda, K.: Discovering Combinatorial Interactions in Survival Data. Bioinformatics, 2013 Sep 13, [Epub ahead of print] (PMID: 24037215)

J015. Eikawa, S., Kakimi, K., Isobe, M., Kuzushima, K., Luescher, I., Ohue, Y., Ikeuchi, K., Uenaka, A., Nishikawa, H., Udono, H., Oka, M., Nakayama, E.: Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide. Int J Cancer, 132: 345-354, 2013. (PMID: 22729530)

J016. Elshazley, M., Sato, M., Hase, T., Takeyama, Y., Yamashita, R., Yoshida, K., Toyokuni, S., Ishiguro, F., Osada, H., Sekido, Y., Yokoi, K., Usami, N., Shames, D.S., Kondo, M., Gazdar, A.F., Minna, J.D., Hasegawa, Y.: The circadian clock gene BMAL1 is a novel therapeutic target for malignant mesothelioma. Int J Cancer, 131: 2820-2831, 2012. (PMID: 22510946)

J017. Fan, Q., Barathi, V.A., Cheng, C.Y., Zhou, X., Meguro, A., Nakata, I., Khor, C.C., Goh, L.K., Li, Y.J., Lim, W., Ho, C.E., Hawthorne, F., Zheng, Y., Chua, D., Inoko, H., Yamashiro, K., Ohno-Matsui, K., Matsuo, K., Matsuda, F., Vithana, E., Seielstad, M., Mizuki, N., Beuerman, R.W., Tai, E.S., Yoshimura, N., Aung, T., Young, T.L., Wong, T.Y., Teo, Y.Y., Saw, S.M.: Genetic variants on chromosome 1q41 influence ocular axial length and high myopia. PLoS Genet, 8: e1002753, 2012. (PMID: 22685421)

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J022. Garcia_closas, M., Cough, F., Lindstrom, S., Michailidou, K., Schmidt, M.K., Brook, M., Orr, N., Thie, S.K., Riboli, E., Feigelson, H.S., Le Marchand, L., Buring, J.E., Eccles, D., Mrion, P., Fasching, P.A., Bruch, H., Chang-Claude, J., Carpenter, J., Godwin, A., Nevanlinna, H., Giles, G.G., Cox, A., Hopper, J.L., Humphreys, M.K., Wang, Q., Dennis, J., Dicks, E., Howat, W.J., Schoof, N., Bojesen, S.E., Lambrechts, D., Broeks, A., Andrulis, I.L., Guenel, P., Burwinkel, B., Sawyer, E.J., Hollestelle, A., Fletcher, O., Winqvist, R., Brenner, H., Mannermaa, A., Hamann, U., Meindl, A., Lindblom, A., Zheng, W., Devillee, P., Goldberg, M.S., Lubinski, J.,�Kristensene, V., Swerdlow, A., Anton-Culver, H., Dork, T., Muir, K., Matsuo, K., Wu, A.H., Radice, P., Teo, S.H., Shu, X.O., Blot, W., Kang, D., Hartman, M.,�Sangrajrang, S., Shen, C.Y., Sothey, M.C., Park, D.J., Hammet, F., Stone, J., Veer, L.J.V., Rutgers, E.J., Lophatananon, A., Stewart-Brown, S., Siriwanarangsan, P.,�Peto, J ., Schrauder, M.G., Ekici, A.B., Beckmann, M.W., dos Santos

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J110. Okamoto, S., Amaishi, Y., Goto, Y., Ikeda, H., Fujiwara, H., Kuzushima, K., Yasukawa, M., Shiku, H., Mineno, J.: A promising vector for TCR gene therapy: differential effect of siRNA, 2A peptide, and disulfide bond on the introduced TCR expression. Mol Ther Nucleic Acids, 1: e63, 2012. (PMID: 23250361)

J111. Okanami, Y., Tsujimura, K., Mizuno, S., Tabata, M., Isaji, S., Akatsuka, Y., Kuzushima, K., Takahashi, T., Uemoto, S.: Intracellular

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J112. Ono, H., Chihara, D., Chiwaki, F., Sasaki, H., Yanagihara, K., Yoshida, T., Saeki, N., Matsuo, K.: Missense allele of a single nucleotide�polymorphism rs2294008 attenuated anti-tumor effects of prostate stem�cell antigen (PSCA) gene in gallbladder cancer cells. J Carcinog, 12: 4, 2013. (PMID: 23599686)

J113. Ozaki, H., Matsuzaki, H., Ando, H., Kaji, H., Nakanishi, H., Ikehara, Y., Narimatsu, H.: Enhancement of metastatic ability by ectopic expressionof ST6GalNAcI on a gastric cancer cell line in a mouse model. Clin Exp Metastas, 29: 229-238, 2012. (PMID: 22228572)

J114. Ozaki, S., Takigawa, N., Ichihara, E., Hotta, K., Oze, I., Kurimoto, E., Fushimi, S., Ogino, T., Tabata, M., Tanimoto, M., Kiura, K.: Favorable response of heavily treated Wilms' tumor to paclitaxel and carboplatin. Onkologie, 35: 283-286, 2012. (PMID: 22868510)

J115. Oze, I., Matsuo, K., Ito, H., Wakai, K., Nagata, C., Mizoue, T., Tanaka, K., Tsuji, I., Tamakoshi, A., Sasazuki, S., Inoue, M., Tsugane, S.; for the Research Group for the Development and Evaluation of Cancer Prevention Strategies in Japan.: Cigarette smoking and esophageal cancer risk: an evaluation based on a systematic review of epidemiologic evidence among the Japanese population. Jpn J Clin Oncol, 42: 63-73, 2012. (PMID: 22131340)

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J117. Patnode, M., Yu, S-Y., Cheng, C-W., Ho, M-Y., Tegesjö, L., Sakuma, K., Uchimura, K., Khoo, K-H., Kannagi, R., Rosen, S.: KSGal6ST generates galactose-6-O-sulfate in high endothelial venules but does not contribute to L-selectin dependent lymphocyte homing. Glycobiology, 23: 381-394, 2013. (PMID: 23254996)

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J120. Pham, N.M., Mizoue, T., Tanaka, K., Tsuji, I., Tamakoshi, A., Matsuo, K., Ito, H., Wakai, K., Nagata, C., Sasazuki, S., Inoue, M., Tsugane, S.: Physical activity and colorectal cancer risk: an evaluation based on a systematic review of epidemiologic evidence among the Japanese population. Jpn J Clin Oncol, 42: 2-13, 2012. (PMID: 22068300)

J121. Pharoah, P.D., Tsai, Y.Y., Ramus, S.J., Phelan, C.M., Goode, E.L., Lawrenson, K., Buckley, M., Fridley, B.L., Tyrer, J.P., Shen, H., Weber, R., Karevan, R., Larson, M.C., Song, H., Tessier, D.C., Bacot, F., Vincent, D., Cunningham, J.M., Dennis, J., Dicks, E; Australian Cancer Study; Australian Ovarian Cancer Study Group, Aben, K.K., Anton-Culver, H., Antonenkova, N., Armasu, S.M., Baglietto, L., Bandera, E.V., Beckmann, M.W., Birrer, M.J., Bloom, G., Bogdanova, N., Brenton, J.D., Brinton, L.A., Brooks-Wilson, A., Brown, R., Butzow, R., Campbell, I., Carney, M.E., Carvalho, R.S., Chang-Claude, J., Chen, Y.A., Chen, Z., Chow, W.H., Cicek, M.S., Coetzee, G., Cook, L.S., Cramer, D.W., Cybulski, C., Dansonka-Mieszkowska, A., Despierre, E., Doherty, J.A., Drk, T., du Bois, A., Drst, M., Eccles, D., Edwards, R., Ekici, A.B., Fasching, P.A., Fenstermacher, D., Flanagan, J., Gao, Y.T., Garcia-Closas, M., Gentry-Maharaj, A., Giles, G., Gjyshi, A., Gore, M., Gronwald, J., Guo,

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J122. Saito, K., Sakaguchi, M., Iioka, H., Matsui, M., Nakanishi, H., Huh, N.H., Kondo, E.: Coxsackie and adenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma cells. Oncogene, 2013. Mar 18, doi: 10.1038/onc.2013.66. [Epub ahead of print] (PMID: 23503452)

J123. Saito, K., Takigawa, N., Ohtani, N., Iioka, H., Tomita, Y., Ueda, R., Fukuoka, J., Kuwahara, K., Ichihara, E., Kiura, K., Kondo, E.: Anti-tumor impact of p14ARF on gefitinib-resistant non-small cell lung cancers. Mol Cancer Ther, 12:1616-1628, 2013. (PMID: 23761220)

J124. Saito, S., Murata, T., Kanda, T., Isomura, H., Narita, Y., Sugimoto, A., Kawashima, D., Tsurumi, T.: Epstein-Barr virus deubiquitinase downregulates TRAF6-mediated NF-κB signaling during productive replication. J Virol, 87: 4060-4070, 2013. (PMID: 23365429)

J125. Sakuma, K., Aoki, M., Kannagi, R.: Transcription factors c-Myc and CDX2 mediate

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J126. Sakuma, K., Chen, G.Y., Aoki, M., Kannagi, R.: Induction of 6-sulfated glycans with cell adhesion activity via T-bet and GATA-3 in human helper T cells. Biochem Biophys Acta, 1820: 841-848, 2012. (PMID: 23088960)

J127. Sakuma, K., Furuhashi, T., Kondo, S., Yabe, U., Ohmori, K., Ito, H., Aoki, M., Morita, A., Kannagi, R.: Sialic acid cyclization of human Th homing receptor glycan associated with recurrent exacerbations of atopic dermatitis. J Dermatol Sci, 68: 187-193, 2012. (PMID: 23088960)

J128. Sasazuki, S., Charvat, H., Hara, A., Wakai, K., Nagata, C., Nakamura, K., Tsuji, I., Sugawara, Y., Tamakoshi, A., Matsuo, K., Oze, I., Mizoue, T., Tanaka, K., Inoue, M., Tsugane, S.. Diabetes mellitus and cancer risk: Pooled analysis of eight cohort studies in Japan. Cancer Sci, 2013, Jul 26. doi: 10.1111/cas.12241. [Epub ahead of print] (PMID:23889822)

J129. Sato, F., Sawamura, M., Ojima, M., Tanaka, K., Hanioka, T., Tanaka, H., Matsuo, K.: Smoking increases risk of tooth loss: A meta-analysis of the literature. World J Meta-analysis, 1: 16-26, 2013.

J130. Sekido Y.: Molecular genetics of malignant mesothelioma. eLS, WILEY -ONLINE LIBRARY, 2012. Published Online: 16 JUL 2012.

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J133. Shen, H., Fridley, B.L., Song, H., Lawrenson, K., Cunningham, J.M., Ramus, S.J., Cicek, M.S., Tyrer, J., Stram, D., Larson, M.C., Kbel, M; PRACTICAL Consortium, Ziogas, A., Zheng, W., Yang, H.P., Wu, A.H., Wozniak, E.L., Ling, Woo, Y., Winterhoff, B., Wik, E., Whittemore, A.S., Wentzensen, N., Palmieri, Weber, R., Vitonis, A.F., Vincent, D., Vierkant, R.A., Vergote, I., Van Den Berg, D., Van Altena, A.M., Tworoger, S.S., Thompson, P.J., Tessier, D.C., Terry, K.L., Teo, S.H., Templeman, C., Stram, D.O., Southey, M.C., Sieh, W., Siddiqui, N., Shvetsov, Y.B., Shu, X.O.,

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J139. Shitara, K., Matsuo, K., Muro, K., Doi, T., Ohtsu, A.: Progression-free survival and�post-progression survival in patients with advanced gastric cancer treated with�first -line chemotherapy. J Cancer Res Clin, 139: 1383-1389, 2013. (PMID: 23708301)

J140. Shitara, K., Sawaki, A., Matsuo, K., Kondo, C., Takahari, D., Ura, T., Tajika, M., Niwa, Y., Muro, K.: A retrospective comparison of S-1 plus cisplatin and capecitabine plus cisplatin for patients with advanced or recurrent gastric cancer. Int J Clin Oncol, 18: 539-546, 2013. (PMID: 22552360)

J141. Shitara, K., Yatabe, Y., Matsuo, K., Sugano, M., Kondo, C., Takahari, D., Ura, T., Tajika, M., Ito, S., Muro, K.: Prognosis of patients with advanced gastric cancer by HER2 status and trastuzumab treatment. Gastric Cancer, 16: 261-267, 2013. (PMID: 22797858)

J142. Shitara, K., Yuki, S., Tahahari, D., Nakamura, M., Kondo, C., Tsuda, T., Kii, T., Tsuji, Y., Utsunomiya, S., Ichikawa, D., Hosokawa, A., Ishiguro, A., Sakai, D., Hironaka, S., Oze, I., Matsuo, K., Muro, K. Randomised phase II study comparing dose-escalated weekly paclitaxel vs

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standard-dose weekly paclitaxel for patients with previously treated advanced gastric cancer. Brit J Cancer, 110: 271-277, 2014. (PMID:24281004)

J143. Suda, K., Tomizawa, K., Osada, H., Maehara, Y., Yatabe, Y., Sekido, Y., Mitsudomi, T.: Conversion from the “oncogene addiction” to “drug addiction” by intensive inhibition of the EGFR and MET in lung cancer with activating EGFR mutation. Lung cancer, 76: 292-299, 2012. (PMID: 22133747)

J144. Sueta, A., Ito, H., Islam, T., Hosono, S., Watanabe, M., Hirose, K., Fujita, T., Yatabe, Y., Iwata, H., Tajima, K., Tanaka, H., Matsuo, K.: Differential impact of body mass index and its change on the risk of breast cancer by molecular subtype: A case-control study in Japanese women. SpringerPlus, 1: 39, 2012

J145. Sueta, A., Ito, H., Kawase, T., Hirose, K., Hosono, S., Yatabe, Y., Tajima, K., Tanaka, H., Iwata, H., Iwase, H., Matsuo, K.: A genetic risk predictor for breast cancer using a combination of low-penetrance polymorphisms in a Japanese population. Breast Cancer Res Tr, 132: 711-721, 2012. (PMID: 22160591)

J146. Sugimoto, A., Sato, Y., Kanda, T., Murata, T., Narita, Y., Kawashima, D., Kimura, H., Tsurumi, T.: Different distributions of Epstein-Barr virus early and late gene transcripts within viral replication compartments. J Virol, 87: 6693-6699, 2013. (PMID: 23552415)

J147. Taguchi, O., Tsujimura, K., Kontani, K., Harada, Y., Nomura, S., Ikeda, H., Morita, A., Sugiura, H., Hayashi, N., Yatabe, Y., Seto, M., Tatematsu, M., Takahashi, T., Fukushima, A.: Behavior of bone marrow-derived cells following in vivo transplantation: Differentiation into stromal cells with roles in organ maintenance. Am J Pathol, 1482: 1255-1262, 2013. (PMID: 23416163)

J148. Takagi, M., Ueda, J., Hwang, J-H., Hashimoto, J., Izumikawa, M., Murakami, H., Sekido, Y., Shin-ya, K.: Tyrosyl-DNA phosphodiesterase 1 inhibitor from an anamorphic fungus. J Nat Prod, 75: 764-767, 2012. (PMID: 22390627)

J149. Tamanaka, T., Oka, Y., Fujiki, F., Tsuboi, A., Katsuhara, A., Nakajima, H., Hosen, N., Nishida, S., Lin, Y.H., Tachino, S., Akatsuka, Y., Kuzushima, K., Oji, Y., Kumanogoh, A., Sugiyama, H.: Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor. Anticancer Res, 32: 5201-5209, 2012. (PMID: 23225417)

J150. Tanaka, I., Osada, H., Fujii, M., Fukatsu, A., Hida, T., Horio, Y., Kondo, Y., Sato, A., Hasegawa, Y., Tsujimura, T., Sekido, Y.: LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade. Oncogene, 2013 Dec 16, [Epub ahead of print] (PMID: 24336325)

J151. Tanaka, K., Tamiya-Koizumi, K., Hagiwara, K., Ito, H., Takagi, A., Kojima, T., Suzuki, M., Iwaki, S., Fujii, S., Nakamura, M., Banno, Y., Kannagi, R., Tsurumi, T., Kyogashima, M., Murate, T.: Role of down-regulated neutral ceramidase during all-trans retinoic acid-induced neuronal differentiation in SH-SY5Y neuroblastoma cells. J Biochem, 151: 611-6200, 2012. (PMID: 22451680)

J152. Tanaka, M., Ma, E., Tanaka, H., Ioka, A., Nakahara, T., Takahashi, H.: Trends of stomach cancer mortality in eastern Asia in 1950-2004: Comparative study of Japan, Hong Kong and Singapore using age, period and cohort analysis. Int J Cancer, 130: 930-936, 2012. (PMID: 21425256)

J153. Taniguchi, C., Tanaka, H., Oze, I., Ito, H., Saka, H., Tachibana, K., Tokoro, A., Nozaki, Y., Nakamichi, N., Suzuki, Y., Suehisa, H., Sakakibara, H.: Factors associated with weight gain after smoking cessation therapy in Japan. Nurs Res, 62: 414-421, 2013. (PMID: 24165217)

J154. Tanikawa, C., Matsuo, K., Kubo, M., Takahashi, A., Ito, H., Tanaka, H., Yatabe, Y., Yamao, K., Kamatani, N., Tajima, K., Nakamura, Y., Matsuda, K.: Impact of PSCA variation on gastric ulcer susceptibility. Plos One, 8: e63698, 2013. (PMID: 23704932)

J155. Tcherniuk, S., Fiser, A-L., Derouazi, M., Toussaint, B., Wang, Y., Wojtal, I., Kondo, E., Szolajska, E., Chroboczek, J.: Certain protein transducing agents convert translocated proteins into cell killers. Acta Biochim Pol, 59: 433-439, 2012. (PMID: 22946029)

J156. Terao, C., Ohmura, K., Ikari, K., Kochi, Y., Maruya, E., Katayama, M., Yurugi, K., Shimada, K., Murasawa, A., Honjo, S., Takasugi, K., Matsuo, K., Tajima, K., Suzuki, A., Yamamoto, K., Momohara, S., Yamanaka, H., Yamada, R., Saji, H., Matsuda, F., Mimori, T.: ACPA-negative RA consists of two genetically distinct subsets based on RF positivity in Japanese. Plos One, 7: e40067, 2012. (PMID: 22792215)

J157. Teshima, K., Nara, M., Watanabe, A., Ito, M., Ikeda, S., Hatano, Y., Oshima, K., Seto, M., Sawada, K., Tagawa, H.: Dysregulation of BMI1

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and microRNA-16 collaborate to enhance an anti-apoptotic potential in the side population of refractory mantle cell lymphoma. Oncogene, 2013. May 20. doi: 10.1038/onc.2013.177. [Epub ahead of print] (PMID: 23686310)

J158. Toda, M., Kuo, C.H., Borman, S.K., Richardson, R.M., Inoko, A., Inagaki, M., Collins, A., Schneider, K., Ono, S.J.: Evidence that formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) complex mediates mast cell activation following Fc{epsilon}RI/CC chemokine receptor 1 cross-talk. J Biol Chem, 287: 24516-24542, 2012. (PMID: 22613718)

J159. Tokunaga, T., Shimada, K., Yamamoto, K., Chihara, D., Ichihashi, T., Oshima, R., Tanimoto, M., Iwasaki, T., Isoda, A., Sakai, A., Kobayashi, H., Kitamura, K., Matsue, K., Taniwaki, M., Tamashima, S., Saburi, Y., Masunari, T., Naoe, T., Nakamura, S., Kinoshita, T.: Retrospective analysis of prognostic factors for angioimmunoblastic T-cell lymphoma: a multicenter cooperative study in Japan. Blood, 119: 2837-2843, 2012. (PMID: 22308294)

J160. Tsuzuki, S., Seto, M.: Expansion of functionally defined mouse hematopoietic stem and progenitor cells by a short isoform of RUNX1/AML1. Blood, 119: 727-735, 2012. (PMID: 22130803)

J161. Tsuzuki, S., Seto, M.: TEL(ETV6)-AML1(RUNX1) initiates self-renewing fetal pro-B cells in association with a transcriptional program shared with embryonic stem cells in mice. Stem Cells, 36: 236-247, 2013. (PMID: 23135987)

J162. Uemura, H., Hiyoshi, M., Arisawa, K., Yamaguchi, M., Naito, M., Kawai, S., Hamajima, N., Matsuo, K., Taguchi, N., Takashima, N., Suzuki, S., Hirasada, K., Mikami, H., Ohnaka, K., Yoshikawa, A., Kubo, M., Tanaka, H.: Japan Multi-institutional Collaborative Cohort.: Gene variants in PPARD and PPARGC1A are associated with timing of natural menopause in the general Japanese population. Maturitas, 71: 369-375, 2012. (PMID: 22310107)

J163. Ueno, T., Toyooka, S., Suda, K., Soh, J., Yatabe, Y., Miyoshi, S., Matsuo, K., Mitsudomi, T.: Impact of age on epidermal growth factor receptor mutation in lung cancer. Lung Cancer, 78: 207-211, 2012. (PMID: 23036155)

J164. Umino, A., Seto, M.: Array CGH reveals clonal evolution of adult T-cell leukemia/lymphoma. Methods Mol Biol, 973: 189-196, 2013. (PMID:

23412791)

J165. Yamada, T., Takeuchi, S., Fujita, N., Nakamura, A., Wang, W., Li, Q., Oda, M., Mitsudomi, T., Yatabe, Y., Sekido, Y., Yoshida, J., Higashiyama, M., Noguchi, M., Uehara, H., Nishioka, Y., Sone, S., Yano, S.: Akt kinase-interacting protein1, a novel therapeutic target for lung cancer with EGFR-activating and gatekeeper mutations. Oncogene, 32: 4427-4435, 2013. (PMID: 23045273)

J166. Yamaguchi, T., Yanagisawa, K., Sugiyama, R., Hosono, Y., Shimada, Y., Arima, C., Kato, S., Tomida, S., Suzuki, M., Osada, H., Takahashi, T.: NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma. Cancer Cell, 21: 348-361, 2012 (PMID: 22439932)

J167. Yamamoto, K., Tsuzuki, S., Minami, Y., Yamamoto, Y., Abe, A., Ohshima, K., Seto, M., Naoe, T.: Functionally deregulated AML1/RUNX1 cooperates with BCR-ABL to induce a blastic phase-like phenotype of chronic myelogenous leukemia in mice. Plos One, 8: e74864, 2013. (PMID: 24098673)

J168. Yamamura, T., Hikita, J., Bleakley, M., Hirosawa, T., Sato-Otsubo, A., Torikai, H., Hamajima, T., Nannya, Y., Demachi-Okamura, A., Maruya, E., Saji, H., Yamamoto, Y., Takahashi, T., Emi, N., Morishima, Y., Kodera, Y., Kuzushima, K., Riddell, S.R., Ogawa, S., Akatsuka, Y.: HapMap SNP Scanner: an online program to mine SNPs responsible for cell phenotype. Tissue Antigens, 80: 119-125, 2012. (PMID: 22568758)

J169. Yamashita, Y., Ito, Y., Isomura, H., Takemura, N., Okamoto, A., Motomura, K., Tsujiuchi, T., Natsume, A., Wakabayashi, T., Toyokuni, S., Tsurumi, T.: Lack of presence of the human cytomegalovirus in human glioblastoma. Mod Pathol, 2013. Epub ahead of print. (PMID: 24336154)

J170. Yanagisawa, K., Tomida, S., Matsuo, K., Arima, C., Kusumegi, M., Yokoyama, Y., Ko, S.B., Mizuno, N., Kawahara, T., Kuroyanagi, Y., Takeuchi, T., Goto, H., Yamao, K., Nagino, M., Tajima, K., Takahashi, T.: Seven-signal proteomic signature for detection of operable pancreatic ductal adenocarcinoma and their discrimination from autoimmune pancreatitis. Int J Proteomics, 2012: 510397, 2012. (PMID: 22675630)

J171. Yokobori, T., Iinuma, H., Shimamura, T., Imoto, S., Sugimachi, K., Ishii, H., Iwatsuki, M., Ota, D., Ohkuma, M., Iwaya, T., Nishida, N., Kogo,

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J172. R., Sudo, T., Tanaka, F., Shibata, K., Toh, H., Sato, T., Barnard, G.F., Fukagawa, T., Yamamoto, S., Nakanishi, H., Sasaki, S., Miyano, S., Watanabe, T., Kuwano, H., Mimori, K., Pantel, K., Mori, M.: Plastin 3 is a novel marker for circulating tumor cells undergoing the epithelial–mesenchymal transition and is associated with colorectal cancer prognosis. Cancer Res, 73: 2059-2069, 2013. (PMID: 23378342)

J173. Yoshida, N., Nishikori, M., Izumi, T., Imaizumi, Y., Sawayama, Y., Niino, D., Tashima, M., Hoshi, S., Ohshima, K., Shimoyama, M., Seto, M., Tsukasaki, K.: Primary peripheral T-cell lymphoma, not otherwise: specified of the thyroid with autoimmune thyroiditis. Brit J Haematol, 161: 214-223, 2013. (PMID: 23432459)

J174. Yoshida, N., Oda, M., Kuroda, Y., Katayama, Y., Okikawa, Y., Masunari, T., Fujiwara, M., Nishisaka, T., Sasaki, N., Sadahira, Y., Mihara, K., Asaoku, H., Matsui, H., Seto, M., Kimura, A., Arihiro, K., Sakai, A.: Clinical Significance of sIL-2R Levels in B-Cell Lymphomas. PLoS One. 8: e78730. 2013. (PMID: 24236041)

J175. Yoshida, N., Umino, A., Liu, F., Arita, K., Karube, K., Tsuzuki, S., Ohshima, K., Seto, M.: Identification of multiple subclones in peripheral T-cell lymphoma, not otherwise specified with genomic aberrations. Cancer Medicine, 1: 289-294, 2012. (PMID: 23342278)

J176. Yoshioka, S., Tsukamoto, Y., Hijiya, N., Nakada, C., Uchida, T., Matsuura, K., Takeuchi, I., Seto, M., Kawano, K., Moriyama, M.: Genomic profiling of oral squamous cell carcinoma by array-based comparative genomic hybridization. PLoS One, 8: e56165, 2013. (PMID: 23457519)

J177. Yuasa, Y., Nagasaki, H., Oze, I., Akiyama, Y., Yoshida, S., Shitara, K., Ito, S., Hosono, S., Watanabe, M., Ito, H., Tanaka, H., Kang, D., Pan, K.F., You, W.C., Matsuo, K.: Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk. Int J Cancer, 131: 2596-2603, 2012. (PMID: 22447362)

J178. Zheng, W., Zhang, B., Cai, Q., Sung, H., Michailidou, K., Shi, J., Cho, J.Y., Long, J., Dennis, J., Hymphreys, M.K., Wang, Q., Lu, W., Gao, Y.T., Li, C., Cai, F., Park, S., Yoo, K.Y., Noh, D.Y., Han, W., Dunning, A.M., Benitez, J., Vincent, D., Bacot, F., Tessier, D., Kim, S.W., Le, M.H., Lee, J.W., Lee, J.Y., Xiang, Y.B., Zheng, Y., Wang, W., Ji, B.T., Matsuo, K., Ito, H., Iwata, H., Tanaka, H., Wu, A.H., Tseng, C.C., van den Berg, D., Stram, D.O., Teo, S.H., Yip, C.H., Kang, I.N., Wong, T.Y.,

Shen, C.Y., Yu, J.C., Huang, C.S., Hous, M.F., Hartman, M., Miao, H., Lee, S.C., Putti, T.C., Muir, K., Laphatananon, A., Stewart-Brown, S., Siriwanarangsan, P., Sangrajrang, S., Shen, H., Chen, K., Wu, P.E., Ren, Z., haiman, C.A., Sueta, A., Kim, M., Khoo, U.S., Iwasaki, M., Pharaoah, P.D.P., Wen, Q., Hall, P., Shu, X.O., Easton, D.F., Kang, D.: Common genetic determinants of breast-cancer risk in East Asian women: a collaborative study of 23,637 breast cancer cases and 25,579 controls. Hum Mol Genet, 22: 2539-2550, 2013. (PMID: 23535825)

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Reviews and Books R001. Fujii, M., Nakanishi, H., Toyoda, T., Tanaka, I., Kondo, Y., Osada, H., Sekido, Y.: Convergent signaling in the regulation of the connective tissue growth factor in malignant mesothelioma: TGF- signaling and defects in the Hippo signaling cascade. Cell Cycle, 11: 3373-3379, 2012. (PMID: 22918238)

R002. Fujii, M.: Exploration of a new drug that targets YAP. J Biochem, 152: 209-211, 2012. (PMID: 22761457)

R003. Goto, H., Inagaki, M.: Method for generation of antibodies specific for site-and post-translational modifications. Monoclonal Antibodies: Methods and Protocols, Methods in Molecular Biology, vol. 1131, Springer Science+Business Media New York 2014. in press.

R004. Goto, H., Inoko, A., Inagaki, M.: Cell cycle progression by the repression of primary cilia formation in proliferating cells. Cell Mol Life Sci, 70: 3893-3905, 2013. (PMID: 23475109)

R005. Goto, H., Izawa, I., Li, P., Inagaki, M.: Novel regulation of checkpoint kinase 1: Is checkpoint kinase 1 a good candidate for anti-cancer therapy? Cancer Sci, 103: 1195-1200, 2012. (PMID: 22435685)

R006. Murata, T., Tsurumi, T.: Epigenetic modification of the Epstein-Barr virus BZLF1 promoter regulates viral reactivation from latency. Front Genet, 4: 53, 2013. (PMID: 23577022)

R007. Murata, T., Tsurumi, T.: Switching of EBV cycles between latent and lytic states. Rev Med Virol, Epub ahead of print, 2013. (PMID: 24339346)

R008. Ohmuro-Matsuyama, Y., Inagaki, M., Ueda, H.: Detection of protein phosphorylation by open-sandwich immunoassay. Integrative Proteomics, ed. Leung, H.-C.E. InTech, 197-214, 2012.

R009. Okamoto, Y., Kondo, Y.: Genetic and epigenetic alterations in inflammation-related cancers - General Mechanisms of Cancers. From Inflammation to Cancer, 29-48, 2012.

R010. Sato, Y., Tsurumi, T.: Genome guardian p53 and viral infections. Rev Med Virol, 23: 213-220, 2012. (PMID: 23255396)

R011. Sekido, Y.: Molecular pathogenesis of malignant mesothelioma. Carcinogenesis, 34:

1413-1419, 2013. (PMID: 23677068)

R012. Shinjo, K., Kondo, Y.: Clinical implications of epigenetic alterations in human thoracic malignancies: epigenetic alterations in lung cancer. Methods Mol Biol, 221-239, 2012. (PMID: 22359296)

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Abstracts for international conferences

A001. Akatsuka, Y., Taji, H., Morishima, Y., Miyamura, K., Kodera, Y., Emi, N., Takahashi, T., Kinoshita, T., Kuzushima, K.: Vaccination with minor histocompatibility antigen-derived peptides in post-transplant patients with hematological malignancies - preliminary results. 2nd International Workshop Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation, Bethesda, 2012.

A002. Arita, K., Seto, M.: New mouse models of B-cell lymphoma using in vitro retroviral transduction system. 9th AACR/JCA Joint Conference, Hawaii, 2013.

A003. Chihara, D., Asano, N., Kinoshita, T., Maeda, Y., Matsue, K., Ohmachi, K., Okamoto, M., Mizuno, I., Uchida, T., Nagai, H., Ogura, M., Suzuki, R.: Simplified MIPI is a valid prognostic index in the rituximab era: Multicenter MCL study in Japan. Blood Abstracts 55th Annual Meeting, New Orleans, 2013.

A004. Chihara, D., Ito, H., Matsuda, T., Katanoda, K., Shibata, A., Saika, K., Sobue, T., Matsuo, K.: Decreasing trend in mortality of adult T-cell Leukemia/Lymphoma in Japan. T-cell Lymphoma Forum, San Francisco, 2013

A005. Chihara, D., Izutsu, K., Kondo, E., Sakai, R., Mizuta, S., Yokoyama, K., Kaneko, H., Kato, K., Sakamaki, H., Suzuki, R., Suzumiya, J.: High-dose chemotherapy with autologous stem cell transplantation for elderly patients with relapsed/ refractory diffuse large b-cell lymphoma. 12th International onference of Malignant Lymphoma, Switzerland, 2013.

A006. Chihara, D., Morton, L., Ito, H., Weisenburger, T., Matsuo, K.: The difference in the incidence and the trend of States. American Association for Cancer Research Annual Meeting 2013, Washington, 2013.

A007. Fujii, M., Nakanishi, H., Toyoda, T., Tanaka, I., Kondo, Y., Osada, H., Sekido, Y.: Convergent signaling in the regulatuion of malignant mesothelioma growth through CTGF: TGF-β signaling and defects in the Hippo signaling pathway. Ninth AACR-Japanese Cancer Association Joint Conference, Hawaii, 2012.

A008. Fujii, M., Toyoda, T., Nakanishi, H., Yatabe, Y., Sato, A., Hida, T., Tsujimura, T.,

Osada, H., Sekido, Y.: TGF-β synergizes with defects in the Hippo pathway by inducing CTGF expression. AACR Annual Meeting 2012, Chicago, 2012.

A009. Fukumoto, K., Ito, H., Park, C., Tanaka, H., Matsuo, K., Tajima, K., Takezaki, T.: Cigarette smoke inhalation and lung cancer risk: Case-control study in Japan. 12th Annual AACR International Conference on Frontiers in Cancer Prevention Research, Washington, 2013.

A010. Goto, H., Kasahara, K., Izawa, I., Kiyono, T., Watanabe, N., Elowe, S., Nigg, E.A., Inagaki, M.: Novel mitotic signalling crosstalk between PI3K-Akt pathway and Plk1. the 52nd Annual Meeting of the American Society for Cell Biology, San Francisco, 2012.

A011. Goto, H.: Novel mitotic signaling crosstalk between PI3K-Akt pathway and Plk1. 1st International Symposium on Protein Modifications in Pathogenic Dysregulation of Signaling (supported by Grant-in-Aid for Scientific Research on Innovative Area from MEXT), Tokyo, 2013.

A012. Hirosawa, N., Sakamoto, T., Uemura, Y., Sakamoto, Y.: Proteomic analyses of adrenal and pituitary gland of rat exposed fenitrothion. The 26th Annual Symposium of The Protein Society, San Diego, 2012.

A013. Inagaki, M., Goto, H.: Novel regulation of checkpoint kinase 1 (Chk1): Is Chk1 a good candidate for anti-cancer therapy? Mini-Symposium on “Stress Signals & Responses”, Abo Akademi University Center of Excellence “Cell stress and Molecular Aging”, Turku, 2012.

A014. Inagaki, M.: Intermediate filaments and site- and phosphorylation state-specific antibodies. Lecture at the University of Gothenburg, Gothenburg, 2012.

A015. Inagaki, M.: Intermediate filaments and site- and phosphorylation state-specific antibodies. Global COE the 4th International Symposium, Nagoya, 2012.

A016. Inagaki, M.: Pathophysiological roles of intermediate filaments and intermediate filament phosphorylation. International Meeting of the German Society for Cell Biology, Leipzig, 2012.

A017. Inoko, A., Matsuyama, M., Goto, H., Ohmuro-Matsuyama, Y., Hayashi, Y., Enomoto, M., Ibi, M., Urano, T., Yonemura, S., Kiyono, T., Izawa, I., Inagaki, M.: Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells. The 12th biennial Gordon

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Conference on Intermediate Filaments, Lewiston, 2012.

A018. Ito, H., Gallus, S., Hosono, S., Oze, I., Fukumoto, K., Yatabe, Y., Hida, T., Mitsudomi, T., Negri, E., Vecchia, C., Tanaka, H., Matsuo, K.: Time to first cigarette and lung cancer risk in Japan. 12th Annual AACR International Conference on Frontiers in Cancer Prevention Research, Washington, 2013.

A019. Ito, H., Sueta, A., Iwata, H., Hosono, S., Oze, I., Watanabe, M., Iwase, H., Tanaka, H., Matsuo, K.: A genetic risk predictor for breast cancer using a combination of low-penetrance polymorphisms in a Japanese population. An AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment. Hollywood, 2012.

A020. Kanda, T., Murata, T., Tsurumi, T.: Chromosome binding of Epstein-Barr virus EBNA1 protein is mediated by arginine residues within chromosome binding domains. International Congress on Oncogenic Herpesviruses and Associated Diseases, Philadelphia, 2012.

A021. Kanda, T., Murata, T., Tsurumi, T.: Roles of BART microRNAs in EBV-infected epithelial cells. 6th International Symposium on Nasophjaryngeal Carcinoma, Istanbul, 2013.

A022. Kasahara, K., Goshima, N., Matsuzaki, F., Inagaki, M.: Emarging role of the ubiquitin-proteasome pathway in primary cilia assembly. The 25th CDB meeting “Cilia and Centrosomes, from Fertilization to Cancer”, Kobe, 2013.

A023. Kasahara, K., Inagaki, M.: Complex formation between Plk1 and 14-3-3 gamma is essential for metapahse to anaphase transition. Mini-Symposium on “Stress Signals & Responses”, Abo Akademi University Center of Excellence “Cell stress and Molecular Aging”, Turku, 2012.

A024. Kasahara, K., Inagaki, M.: Novel mitotic signaling crosstalk between PI3K-Akt pahtway and Plk1. Seminar for Center for Brain Repair and Rehabilitation, Gothenburg, 2012.

A025. Kasahara, K.: Novel mitotic signalling crosstalk between PI3K-Akt pathway and Plk1. Global COE the 4th International Symposium, Nagoya, 2012.

A026. Katsushima, K., Shinjo, K., Natsume, A., Ohka, F., Fujii, M., Osada, H., Sekido, Y., Kondo, Y.: Contribution of microRNA-1275 to Claudin11

suppression via polycomb-mediated silencing mechanism in human glioma stem-like cells. JSPS Sapporo Cancer Epigenetics Seminar of the A3 Foresight Program 2012, Sapporo, 2012.

A027. Katsushima, K., Shinjo, K., Ohka, F., Fujii, M., Osada, H., Sekido, Y., Natsume, A., Kondo, Y.: Epigenetic regulation of miR-1275 through histone H3 lysine 27 trimethylation during human glioma stem-like cell differentiation. AACR Annual Meeting 2012, Chicago, 2012.

A028. Katsushima, K.: Functional analysis of large non-coding RNA associated with glioma stem cell maintenance. The 18th Korea-Japan Cancer Research Workshop, Gifu, 2013.

A029. Kondo, E.: Development of the Peptide-based anti-tumor DDS technology. Special lecture (invited lecture at Stem Cell and Bioevaluation, WCU Biolodulation Program Animal Biotech Major, Dept Ag Biotech, CALS), Seoul national University, Seoul, 2013

A030. Kondo, Y.: Epigenetic Plasticity and its clinical implications in human neoplasia. Ninth AACR-Japanese Cancer Association Joint Conference, Hawaii, 2013.

A031. Kondo, Y.: Hepatitis virus infection affects DNA methylation in mice with humanized livers. The 18th Korea-Japan Cancer Research Workshop, Gifu, 2013.

A032. Kondo, Y.: Mechanistic link between hepatitis viral infection and induction of aberrant DNA methylation in human hepatocyte chimeric mice. The 17th Japan-Korea Cancer Research Workshop, Busan, 2012.

A033. Kondo, Y.: Molecular links between microenvironmental signals and epigenetic reprogramming processes via long non-coding RNA in glioblastoma. French Japanese Cancer Meeting, Toulouse, 2013.

A034. Kondo, Y.: Polycomb repressive complex 2-mediated epigenetic plasticity contributing to establishment of tissue heterogeneity in glioblastoma. The 3rd Shanghai International Conference of Epigenetics in Development and Diseases, Shanghai, 2012.

A035. Kondo, Y.: Study of aberrant DNA methylation in human hepatocyte chimeric mice. International Symposium on Genetic Regulation and Targeted Therapy of Cancer and 3rd Symposium of A3 Foresight Program, Guangzhou, 2012.

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A036. Kondo, Y.: Translational implications of epigenetic changes in human malignancies. 10th International Conference of the Asian Clinical Oncology Society, Seoul, 2012.

A037. Murakami-Tonami, Y., Kishida, S., Kadomatsu, K.: Inactivation of hSgo1 shows synthetic phenotype to MYCN amplification. Advanced neuroblastoma research 2012. Toronto, 2012.

A038. Murakami-Tonami, Y.: Inactivation of Smc2 shows synergistic lethal response to MYCN amplification by regulating DNA damage response genes transcription in neuroblastoma cells. EMBO conference: The DNA damage response in cell physiology and disease, Greece, 2013.

A039. Murata, T., Noda, C., Kanda, T., Tsurumi, T.: Induction of EBV oncogene LMP1 by AP-2 in NPC cells. 6th International Symposium on Nasopharyngeal Carcinoma, Istanbul, 2013.

A040. Murata, T., Tsurumi, T.: Cis- and trans-elements that affect reactivation of Epstein-Barr virus from latency. International Congress on Oncogenic Herpesviruses and Associated Diseases, Philadelphia, 2012.

A041. Murata, T., Tsurumi, T.: Modification of immune/inflammatory system by EBV and its contribution to cancer. 3rd International Symposium on Carcinogenic Spiral and International Symposium on Tumor Biology in Kanazawa, Kanazawa, 2013.

A042. Nakanishi, H., Saito, T., Kondo, C., Muro, K., Kondo, E.: Sensitivity and resistance of newly-developed high and low HER2 gene-amplified gastric cancer cell lines to trastuzumab and lapatinib. 10th International Gastric Cancer Congress, Verona, 2013.

A043. Nakatsuka, R., Uemura, Y., Matsuoka, Y., Takahashi, M., Iwaki, R., Fujioka, T., Sasaki, Y., Sonoda, Y.: Prospectively isolated PDGFRα and Sca-1 double positive dental pulp-derived mesenchymal stem cell-like cells have different characteristics as compared to PDGFRα and Sca-1 double positive bone marrow-derived mesenchymal stem cells. ISSCR 10th Annual Meeting, Yokohama, 2012.

A044. Narita, Y., Murata, T., Kimura, H., Tsurumi, T.: The Epstein-Barr virus DNA polymerase catalytic subunit BALF5 interacts with Pin1 for efficient productive viral DNA replication. 38th Annual International Herpesvirus Workshop, Grand Rapids, 2013.

Natsume, A., Katsushima, K., Shinjo, K., Ohka, F., Hatanaka, A., Ichimura, N., Kondo, Y.: Epigenetic plasticity regulated by polycomb repressive complex 2 in human glioblastoma. Gordon Research Conference, Lucca, 2013.

A045. Ohka, F., Natsume, A., Katsushima, K., Shinjo, K., Kishida, Y., Motomura, K., Momota, H., Wakabayashi, T., Kondo, Y.: The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma. 10th International Conference of the Asian Clinical Oncology Society, Seoul, 2012.

A046. Ohka, F., Natsume, A., Ichimura, N., Hatanaka, A., Katsushima, K., Shinjo, K., Zong, H., Kondo,Y.: Loss of p53 and Nf1 and subsequent epigenetic alterations in glioblastoma mouse model. Gordon Research Conference, Lucca, 2013.

A047. Ohka, F., Natsume, A., Zong, H., Liu, C., Hatanaka, A., Katsushima, K., Shinjo, K., Wakabayashi, T., Kondo, Y.: Interplay between genetic loss of p53, Nf1 and histone modifications in tumorigenesis of glioblastoma. The 18th Annual Meeting of the Society for Neuro-Oncology, San Francisco, 2013.

A048. Ohka, F.: Loss of p53, Nf1 and subsequent epigenetic alterations during tumor formation of Glioblastoma. JSPS Sapporo Cancer Epigenetics Seminar of the A3 Foresight Program 2013, Sapporo, 2013.

A049. Oze, I., Matsuo, K., Hosono, S., Ito, H., Watanabe, M., Ishioka, K., Ito, S., Tajika, M., Yatabe, Y., Niwa, Y., Yamao, K., Nakamura, S., Tajima, K., Tanaka, H.: The Aldehyde Dehydrogenase 2 (ALDH2) Glu504Lys Polymorphism Interacts with Alcohol Drinking in the Risk of Stomach Cancer. The 4th JCA-AACR Special Joint Conference: The Latest Advances in Gastric Cancer Research. Urayasu, 2013.

A050. Sakuma, K.: c-Myc and CDX2 mediate E-selectin ligand glycan expression in colon cancer cells undergoing EMT. Global COE the 4th International Symposium, Nagoya, 2012.

A051. Sekido, Y., Tanaka, I., Fujii, M., Osada, H.: Hippo signaling cascade alteration in malignant mesothelioma. Keystone Symposia, Monterey, 2013.

A052. Sekido, Y., Tanaka, I., Osada, H., Fujii, M.: Hippo signaling pathway inactivation in malignant mesothelioma cells. iMig (international Mesothelioma interest group) 2012, Boston, 2012.

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A053. Sekido, Y.: Dysregulation of Hippo tumor-suppressive pathway in malignant mesothelioma. 15th International Association for the Study of Lung Cancer, Sydney, 2013.

A054. Sekido, Y.: Molecular Abnormalities and Cell Signaling Dysregulation of Malignant Pleural Mesothelioma. The 17th Congress of the Asian Pacific Society of Respirology, Hong Kong, 2012.

A055. Seto, M.: Genomic alterations in malignant lymphoma and its implication in cancer treatment. The 38th Annual Meeting of Korean Cancer Association, Seoul, 2012.

A056. Seto, M.: Meet the Professor Sessions· XV. Malignant lymphoma as a consequence of clonal evolution. 12th International Conference on Malignant Lymphoma, Cinema Corso, Lugano (Switzerland), 2013.

A057. Shinjo, K., Okamoto, Y., Takeuchi, I., Fujii, M., Osada, H., Usami, N., Ito, H., Hida, T., Sekido, Y., Kondo, Y.: Integrated analysis of genetic and epigenetic alterations reveals CpG island methylator phenotype associated with distinct clinical characters of lung adenocarcinoma. AACR Annual Meeting 2012, Chicago, 2012.

A058. Shinjo, K., Okamoto, Y., Tanaka, Y., Jean-Pierre Issa, Kondo,Y.: Innate immune system is adequate for induction of DNA methylation after hepatitis viral infection in human hepatocyte chimeric mouse . Gordon Research Conference, Lucca, 2013.

A059. Sugimoto, A., Kimura, H., Tsurumi, T.: Epstein-Barr virus genome packaging factors converge at inner part of viral genome storeroom of the BMRF1 core within viral replication compartment. International Congress on Oncogenic Herpesviruses and Associated Diseases, Philadelphia, 2012.

A060. Tanaka, H.: Cost-effectiveness of smoking cessationtherapy in Japan. World cancer congress, Canada, 2012.

A061. Tanaka, Hir., Matsuyama, M., Inoko, A., Kondo, E., Kobori, K., Hayashi, Y., Itohara, S., Izawa, I., Inagaki, M.: Disorder of cytokinesis by defect of mitotic vimentin phosphorylation results in chromosomal instability. The 12th biennial Gordon Conference on Intermediate Filaments, Lewiston, 2012.

A062. Tanaka, Hir., Matsuyama, M., Inoko, A., Kondo, E., Kobori, K., Hayashi, Y., Itohara, S., Izawa, I., Inagaki, M.: Disorder of cytokinesis by defect of mitotic vimentin phosphorylation results

in chromosomal instability. Global COE the 4th International Symposium, Nagoya, 2012.

A063. Tanaka, I., Osada, H., Fujii, M., Sekido, Y.: A LIM protein AJUBA suppresses malignant mesothelioma cell proliflation via Hippo signaling pathway. American Association for Cancer Research （ AACR ） Annual Meeting 2013, Washington, 2013.

A064. Tsurumi, T., Kawashima, D.: Generation of Epstein-Barr virus lacking BVLF1 ORF responsible for late gene transcription. Keystone Symposia on Molecular and Cellular Biology: Advancing Vaccines in the Genomic Era, Rio de Janeiro, 2013.

A065. Tsurumi, T., Kawashima, D.: Nuclear Transport of Epstein-Barr Virus DNA Polymerase is dependent on the BMRF1 Polymerase Processivity Factor and Molecular Chaperone Hsp90. The Biology of Molecular Chaperones: From molecules, organelles and cells to misfolding diseases, Santa Margherita di Pula, 2013.

A066. Tsurumi, T., Kawashima, D.: Nuclear transport of Epstein-Barr virus DNA polymerase is dependent on the BMRF1 polymerase processivity factor and molecular chaperone Hsp90. 18th World Congress on Advances in Oncology and 16th International Symposium on Molecular Medicine, Creta Maris, 2013.

A067. Tsurumi, T., Sugimoto, A.: Epstein-Barr virus genome packaging factors converge in inner genome storerooms of BMRF1 cores within viral replication compartments. The 4th EMBO meeting, Nice, 2012.

A068. Tsurumi, T.: Epstein-Barr virus genome packaging factors converge in inner genome storerooms of BMRF1 cores within viral replication compartments. SGM spring meeting, Manchester, 2013.

A069. Tsurumi, T.: Epstein-Barr virus replication factory. The 11th Awaji International Forum on Infection and Immunity, Awaji, 2012.

A070. Yamada, E., Demachi-Okamura, A., Kondo, S., Maki, H., Zhang, R., Uemura, Y., Suzuki, S., Shibata, K., Kikkawa, F., Kuzushima, K.: Identification of novel ovarian cancer-associated antigens and its epitopes for CTL using an HLA-modified cancer cell line. 9th Joint Conference of the American Association for Cancer Research and the Japanese Cancer Association, Hawaii, 2013.

A071. Yamaguchi, T., Yanagisawa, K., Sugiyama,

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R., Hosono, Y., Shimada, Y., Arima, C., Kato, S., Tomida, S., Suzuki, M., Osada, H., Takahashi, T.: NKX2-1/TITF1/TTF-1-induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma. AACR annual meeting 2012, Chicago, 2012.

A072. Yoshida, N., Karube, K., Utsunomiya, A., Tsukasaki, K., Imaizumi, Y., Taira, N., Uike, N., Umino, A., Arita, K., Suguro, M., Tsuzuki, S., Kinoshita, T., Ohshima, K., Seto, M.: Cell cycle deregulation determines acute transformation in chronic type adult T-cell leukemia/lymphoma. 55th Annual Meeting and Exposition, American Society of Hematology, New Orleans, 2013.

A073. Yoshida, N., Umino, A., Liu, F., Arita, K., Karube, K., Tsuzuki, S., Ohshima, K., Seto, M.: Identification of multiple subclones in peripheral T-cell lymphoma, not otherwise specified with genomic aberrations. 54th Annual Meeting and Exposition, American Society of Hematology, Atlanta, 2012.

A074. Yusa, A., Masuda, T., Yamamoto, S., Niimi, M., Douke, H., Okochi, M., Toneri, M., Ito, S., Honda, H., Arai, F., Nakanishi, H.: Development of rapid isolation device for circulating tumor cells (CTCs) using size-based filtration method and its application for single cell gene expression analysis. 3rd Annual Meeting World CTC, Boston, 2012.

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Record of Seminars ___________________________________________________________

Invited Speakers 2012 Jan. 4 Obuse, C. (Laboratory of Molecular and Cellular Biology, Graduate School of Life Science,

Hokkaido University): Crosstalk between constitutive and facultative heterochromatins in the inactive X chromosome.

Jan. 6 Shuda, M. (Cancer Virology Program, University of Pittsburgh Cancer Institute): Merkel cell

polyomavirus: identification of the 7th human tumor virus inserted into the genome of Merkel cell carcinoma.

Mar. 30 Trembray, M.L. (Goodman Cancer Research Centre, McGill University): Tyrosine protein

phosphatases in cancers: Friends or Foes. Apr. 23 Tamanoi, F. (Department of Microbiology, Immunology & Molecular Genetics, University of

California, Los Angeles): New trends in cancer therapy promoted by chemical biology and nanotechnology.

Sep. 14 Oshima, M. (Division of Genetics, Cancer Research Institute, Kanazawa University): Roles of

inflammatory responses in gastric carcinogenesis. Sep. 28 Sato, Y. (Division of Genetics, Kobe University Graduate School of Medicine):

Non-cell-autonomous tumor progression by mitochondrial dysfunction. Nov. 22 Kurumizaka, H. (Laboratory of Structural Biology, Graduate School of Advanced Science and

Engineering, Waseda University): Chromatin structure as the basis of epigenetics. Nov. 30 Quinlan, R. (Biophysical Sciences Institute, School of Biological and Biomedical Sciences, Durham

University): Growth and Form revisited: Applying the D'Arcy Wentworth Thompson principle to the eye lens.

2013 Mar. 1 Ito, K. (Department of Molecular Bone Biology, Graduate School of Biomedical Sciences, Nagasaki

University): A view on “the Biology of Cancer” through the prism of the transcription factor RUNX3. Mar. 14 Enomoto, A. (Department of Pathology, Nagoya University Graduate School of Medicine):

Functions of Girdin, a downstream effector of Akt signaling, in tumor progression and development of psychiatric disorders.

Mar. 14 Toyoshima, F. (Department of Cell Biology, Institute for Virus Research, Kyoto University):

Signaling network that determines the spindle orientation. Mar. 19 Ebi, H. (Division of Medical Oncology, Cancer Research Institute, Kanazawa University):

Molecularly targeted therapy for lung cancer: comparison with other carcinomas. Mar. 21 Asai, A. (Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of

Shizuoka): Exploring anti-cancer agents with novel mechanisms of action.

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Apr. 10 Sawamoto, K. (Department of Developmental & Regenerative Biology, Nagoya City University

Graduate School of Medical Sciences): Mechanisms of neural regeneration intrinsic to the brain. Apr. 16 Takeda, S. (Department of Radiation Genetics, Kyoto University Graduate School of Medicine):

Analyzing toxic or pharmacological actions of chemical agents using genetic methods. Jul. 26 Kitamura, T. (Department of Developmental and Molecular Biology, Albert Einstein College of

Medicine): The role of chemokine in macrophage-mediated breast cancer metastasis. Aug. 9 Sugiura, R. (Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences,

Kinki University): Elucidating the regulatory mechanisms of the MAP kinase signaling pathway and their application to the drug discovery.

Oct. 2 Cheung, E. (Department of Biochemistry, Genome Institute of Singapore): Transcriptional analyses

of nuclear hormone signaling in cancer cells. Oct. 8 Jinushi, M. (Research Center for Infection-Associated Cancer, Institute for Genetic Medicine,

Hokkaido University): A novel regulatory mechanism for tumor immunity mediated by phagocytosis of apoptotic cells.

Dec. 9 Serfling, E. (Department of Molecular Pathology, Institute of Pathology and Comprehensive Cancer

Center, University of Wurzburg): NFATc1 in lymphocytes: Two ‘genes’ in one locus control immunity and cancerogenesis.

Institute Speakers 2012 Jan. 19 Kojima, Y. (Molecular Pathology): Roles of TGF-β and SDF1 in breast cancer microenvironment. Feb. 16 Kasahara, K. (Biochemistry): Regulation of the mitotic kinase Plk1 by the PI3K/Akt pathway. May. 17 Nakata, S. (Oncological Pathology): Roles of the cancer stem cell marker gene LGR5 in

glioblastoma. Jun. 21 Matsuo, K. (Epidemiology and Prevention): Correlation between ALDH2 polymorphism and various

diseases including cancer: recent updates. Jul. 30 Saito, S. (Virology): The Epstein-Barr virus deubiquitinase BPLF1 promotes viral DNA replication

by blocking TRAF6-mediated activation of the NF-kappaB signaling. Oct. 17 Karube, K. (Molecular Medicine): Molecular pathology of NK-cell neoplasms. Nov. 29 Okamura, A. (Immunology): Exploration of novel tumor antigens using an artificial antigen

presenting cell system. 2013 Jan. 30 Kajino, R. (Molecular Pathology): TAK1 suppresses enterocyte death and intestinal inflammation

77

by preventing ROS production. Feb. 14 Goto, H. (Biochemistry): Perspective of kinases regulating cell cycle progression/checkpoint as

therapeutic targets for cancer. Mar. 14 Murakami, Y. (Molecular Oncology): Inactivation of Smc2 shows synergistic lethal response to

MYCN amplification by regulating DNA repair genes transcription in neuroblastoma cells. May. 16 Ito, H. (Epidemiology and Prevention): Cancer prevention strategies based on genetic risks. Jun. 27 Nakanishi, H. (Oncological Pathology): Development and application of a novel device for capturing

circulating tumor cells. Jun. 28 Matsuo, K. (Epidemiology and Prevention): My fourteen years in Aichi Cancer Center. Nov. 15 Uemura, Y. (Immunology): Invariant NKT cells as a possible tool for cancer immunotherapy. Dec. 18 Kanda, T. (Virology): Mechanisms of latent infection of EB virus and its involvement in epithelial

carcinogenesis.

78

Author index for research reports and publications ________________________________________________________________________________ Akatsuka, Y. 26, J013, J064, J111,

J149, J168, A001 Aoki, M. 36, 37, J125, J126, J127 Arita, K. 21, 22, J004, J052, J053,

J070, J071, J175, A002, A072, A073

Chihara, D. 6, J008, J009, J010, J055, J112, J159, A003, A004, A005, A006

Demachi-Okamura, A.

26, J013, J064, J100, J168, A070

Era, S. 39 Fujii, M. 18, 45, J019, J020, J038,

J039, J075, J084, J102, J150, R001, R002, A007, A008, A026, A027, A051, A052, A057, A063

Fujishita, T. 36, 37, J042 Fujita, M. J064, J100 Fukatsu, A. 18, J020, J150 Fukumoto, K. 7, 9, J021, J043, A009,

A018 Furuya, T. 14, J063 Goto, H. 40, 41, J001, J033, J036,

J054, J067, J080, J098, J099, J170, R003, R004, R005, A010, A011, A013, A017

Hatanaka, A. 44, A044, A046, A047 Hayashi, Y. J036, J080, A017, A061,

A062 He, D. 41 Hiramatsu, K. 26 Hirano, K. J062 Hosono, S. 6, 7, 8, J006, J010, J026,

J031, J032, J040, J041, J043, J044, J047, J057, J058, J059, J074, J076, J078, J079, J093, J094, J107, J116, J118, J121, J133, J138, J144, J145, J177, A018, A019, A049

Ichimura, N. A044, A046 Iioka, H. 13, 14, J122, J123 Inaba, H. 41

Inagaki, M. 39, 40, 41, 42, J033, J036, J046, J054, J067, J080, J098, J104, J158, R003, R004, R005, R008, A010, A013, A014, A015, A016, A017, A022, A023, A024, A061, A062

Inoko, A. 40, 41, J036, J080, J104, J158, R004, A017, A061, A062

Ishiguro, F. 18, J016, J020, J038, J039, J084

Ishioka, K. J078, A049 Islam, T. J040, J041, J144 Ito, H. 6, 7, 8, 9, J006, J008,

J009, J010, J018, J019, J022, J031, J032, J040, J041, J043, J044, J057, J058, J059, J073, J076, J078, J079, J080, J082, J091, J093, J094, J115, J116, J120, J127, J138, J139, J144, J145, J151, J153, J154, J177, J178, A004, A006, A009, A018, A019, A049, A057

Izawa, I. 40, 41, 42, J036, J054, J080, R005, A010, A017, A061, A062

Kakiuchi, T. 22 Kanda, T. 30, 32, 33, 34, J048, J049,

J060, J088, J089, J097, J124, J146, A020, A021, A039

Kannagi, R. 36, J117, J125, J126, J127, J151

Karube, K. 21, 22, J004, J052, J053, J062, J070, J071, J131, J175, A072, A073

Kasahara, K. 39, J054, J067, A010, A022, A023, A024, A025

Kasugai, Y. 22, J004 Katayama, M. 21, 22, J004, J052, J156 Katsushima, K. 44, A026, A027, A028,

A044, A045, A046, A047 Kawakita, D. 6, 8, J057, J058, J059,

J076, J081, J116 Kawamoto, E. 39 Kawase, T. J145

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Kawashima, D. 32, J048, J060, J088, J089, J097, J124, J146, A064, A065, A066

Kobori, K. 40, 41, J080, A061, A062 Kondo, E. 12, 13, 14, 40, J019, J053,

J063, J075, J080, J085, J086, J105, J122, J123, J155, A005, A029, A042, A061, A062

Kondo, S. (IM) J064, J127, A070 Kondo, S. (MP) J127 Kondo, Y. 18, 34, 44, 45, J019, J020,

J038, J039, J084, J088, J150, R001, R009, R012, A007, A026, A027, A030, A031, A032, A033, A034, A035, A036, A044, A045, A046, A047, A057, A058

Kumimoto, H. 49 Kuzushima, K. 26, 27, 28, 34, J005, J013,

J015, J035, J049, J064, J083, J090, J100, J110, J111, J149, J168, A001, A070

Li, P. J067, R005 Maki, H. 27, J064, J100, A005,

A070 Maseki, S. J075, J105 Matsudaira, Y. J019 Matsuo, K. 6, 7, 8, 9, J006, J008,

J009, J010, J011, J012, J017, J018, J021, J022, J025, J028, J031, J032, J034, J037, J040, J041, J043, J044, J045, J047, J050, J051, J056, J057, J058, J059, J061, J065, J066, J068, J072, J073, J076, J077, J078, J079, J081, J082, J091, J093, J094, J095, J096, J108, J109, J112, J115, J116, J118, J119, J120, J128, J129, J133, J134, J135, J136, J137, J138, J139, J140, J141, J142, J144, J145, J154, J156, J162, J163, J170, J177, J178, A004, A006, A009, A019, A043, A049

Matsuyama, M. 40, J036, J067, J080, A017, A061, A062

Morishima, Y. J010, J055, J096, J168, A001

Murakami-Tonami, Y.

J014, A037, A038

Murata, T. 31, 32, 33, 34, J048, J060, J087, J088, J089, J097, J124, J146, R006, R007, A020, A021, A039, A040, A041, A044

Nakanishi, H. 13, 14, J003, J019, J075, J085, J086, J105, J113, J122, J172, R001, A007, A008, A042, A074

Nakao, M. J093, J094 Nakata, S. 15, J023, J029 Narita, Y. 33, J048, J060, J089,

J097, J124, J146, A044 Noda, C. A039 Ohka, F. 44, A026, A027, A044,

A045, A046, A047, A048 Okamoto, Y. 45, R009, A057, A058 Osada, H. 18, 45, J016, J019, J020,

J038, J039, J084, J143, J150, J166, R001, A007, A008, A026, A027, A051, A052, A057, A063, A071

Oze, I. 6, 7, 8, 9, J027, J031, J043, J057, J058, J059, J066, J076, J078, J079, J093, J102, J103, J114, J115, J116, J128, J142, J153, J177, A018, A019, A049

Saito, K. 12, 13, 14, J063, J122, J123

Saito, S. 33, J060, J088, J124 Saito, T. A042 Sakuma, K. 36, J117, J125, J126,

J127, A050 Sato, S. 44, 45 Sato, Y. 32, J146, R010 Sekido, Y. 18, 44, 45, J001, J002,

J007, J016, J019, J020, J030, J038, J039, J084, J092, J130, J143, J148, J150, J165, R001, R011, A007, A008, A026, A027, A051, A052, A053, A054, A057, A063

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Seto, M. 21, 22, 23, J004, J010, J052, J053, J055, J062, J070, J071, J131, J132, J147, J157, J160, J161, J164, J167, J173, J174, J175, J176, A002, A038, A055, A056, A072, A073

Shinjo, K. 18, 44, 45, J020, R012, A026, A027, A044, A045, A046, A047, A057, A058

Shiraishi, K. 26 Sueta, A. J018, J040, J082, J144,

J145, J178, A019 Sugimoto, A. 32, J048, J060, J088,

J089, J097, J124, J146, A059, A067

Tanaka, H. (BC) 40, J080, A061, A062 Tanaka, H. (EP) 6, 7, 8, 9, 14, J010, J024,

J025, J026, J027, J028, J031, J032, J034, J040, J041, J043, J044, J056, J057, J058, J059, J068, J075, J076, J078, J079, J080, J085, J086, J093, J094, J101, J105, J106, J107, J108, J109, J116, J129, J138, J144, J145, J152, J153, J154, J162, J177, J178, A009, A018, A019, A049, A060, A061, A062

Tanaka, I. 18, J020, J084, J150, R001, A007, A051, A052, A063

Tanigawa, N. 40 Taniguchi, C. 9, J153 Toneri, M. A074 Tsurumi, T. 30, 31, 32, 33, 34, J048,

J049, J060, J087, J088, J089, J097, J124, J146, J151, J169, R006, R007, R010, A020, A021, A039, A040, A041, A044, A059, A064, A065, A066, A067, A068, A069

Tsuzuki, S. 21, 22, 23, J004, J052, J053, J070, J071, J107, J160, J161, J167, J175, A072, A073

Uemura, Y. 27, 28, J064, J099, J100, A012, A043, A070

Wang, Z. J067 Watanabe, M. 8, J031, J032, J040, J041,

J057, J058, J059, J078, J079, J093, J094, J116, J138, J144, J177, A019, A049

Yamada, E. A070 Yoshida, N. 21, 22, J004, J052, J053,

J070, J071, J131, J173, J174, J175, A072, A073

Yusa, A. A074 Zhang, R. 27, 28, J100, A070