Details of a Researcher - Hamamoto, Junko

Papers - Hamamoto, Junko

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Effects of the common polymorphism in the human aldehyde dehydrogenase 2 (ALDH2) gene on the lung.

Aoi Kuroda, Ahmed E Hegab, Gao Jingtao, Shuji Yamashita, Nobuyuki Hizawa, Tohru Sakamoto, Hideyasu Yamada, Satoshi Suzuki, Makoto Ishii, Ho Namkoong, Takanori Asakura, Mari Ozaki, Hiroyuki Yasuda, Junko Hamamoto, Shizuko Kagawa, Kenzo Soejima, Tomoko Betsuyaku

Respiratory research 18 ( 1 ) 69 - 69 2017.04

ISSN 1465-9921

BACKGROUND: Aldehyde dehydrogenases (ALDHs) play a major role in detoxification of aldehydes. High expression of ALDHs is a marker for stem cells of many organs including the lungs. A common polymorphism in ALDH2 gene (ALDH2*2) results in inactivation of the enzyme and is associated with alcohol flushing syndrome and increased risk for cardiovascular and Alzheimer's diseases and some cancers. The effect of this ALDH2 polymorphism on the lung and its stem cells has not been thoroughly examined. METHODS: We examined the association between the ALDH2*2 allele and lung function parameters in a population of healthy individuals. We also examined its association with the incidence of asthma and COPD in patient cohorts. We used the in vitro colony forming assay to detect the effect of the polymorphism on lung epithelial stem cells from both primary human surgical samples and Aldh2*2 transgenic (Tg) and Aldh2 -/- mice. Response to acute and chronic lung injuries was compared between wild type (WT), Aldh2*2 Tg and Aldh2 -/- mice. RESULTS: In humans, the ALDH2*2 allele was associated with lower FEV1/FVC in the general population, but not with the development of asthma or COPD. Both the bronchial and lung epithelium carrying the ALDH2*2 allele showed a tendency for lower colony forming efficiency (CFE) compared to ALDH2 allele. In mice, the tracheal epithelial thickness, nuclear density, and number of basal stem cells were significantly lower in Aldh2 -/- and Aldh2*2 Tg adult mice than in WT. Electron microscopy showed significantly increased number of morphologically abnormal mitochondria in the trachea of Aldh2 -/- mice. Aldh2 -/- tracheal and lung cells showed higher ROS levels and fewer functional mitochondria than those from WT mice. No significant differences were detected when tracheal and lung epithelial stem cells were examined for their in vitro CFE. When exposed to chronic cigarette smoke, Aldh2*2 Tg mice were resistant to emphysema development, whereas influenza infection caused more epithelial damage in Aldh2 -/- mice than in WT mice. CONCLUSIONS: ALDH2 polymorphism has several subtle effects on the lungs, some of which are similar to changes observed during normal aging, suggesting a "premature lung aging" effect.

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Amplification of EGFR Wild-Type Alleles in Non-Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective EGFR Tyrosine Kinase Inhibitors

Shigenari Nukaga, Hiroyuki Yasuda, Katsuya Tsuchihara, Junko Hamamoto, Keita Masuzawa, Ichiro Kawada, Katsuhiko Naoki, Shingo Matsumoto, Sachiyo Mimaki, Shinnosuke Ikemura, Koichi Goto, Tomoko Betsuyaku, Kenzo Soejima

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 77 ( 8 ) 2078 - 2089 2017.04

ISSN 0008-5472

EGFR-mutated lung cancers account for a significant subgroup of non-small cell lung cancers overall. Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-typeEGFR. Acquired resistance develops to these agents, however, the mechanisms are as yet uncharacterized. In this study, we report that the Src-AKT pathway contributes to acquired resistance to these TKI. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance. These findings underscore the importance of signals from wild-type EGFR alleles in acquiring resistance to mutant-selective EGFR-TKI. Our data provide evidence of wildtype allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. (C) 2017 AACR.

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Overcoming EGFR Bypass Signal-Induced Acquired Resistance to ALK Tyrosine Kinase Inhibitors in ALK-Translocated Lung Cancer

Masayoshi Miyawaki, Hiroyuki Yasuda, Tetsuo Tani, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Shigenari Nukaga, Toshiyuki Hirano, Ichiro Kawada, Katsuhiko Naoki, Yuichiro Hayashi, Tomoko Betsuyaku, Kenzo Soejima

MOLECULAR CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 15 ( 1 ) 106 - 114 2017.01

ISSN 1541-7786

Activation of the EGFR pathway is one of the mechanisms inducing acquired resistance to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib and alectinib. Ceritinib is a highly selective ALK inhibitor and shows promising efficacy in non-small cell lung cancers (NSCLC) harboring the ALK gene rearrangement. However, the precise mechanism underlying acquired resistance to ceritinib is not well-defined. This study set out to clarify the mechanism in ALK-translocated lung cancer and to find the preclinical rationale overcoming EGFR pathway-induced acquired resistance to ALK-TKIs. To this end, ceritinib-resistant cells (H3122-CER) were established from the H3122 NSCLC cell line harboring the ALK gene rearrangement via long-term exposure to ceritinib. H3122-CER cells acquired resistance to ceritinib through EGFR bypass pathway activation. Furthermore, H3122 cells that became resistant to ceritinib or alectinib through EGFR pathway activation showed cross-resistance to other ALK-TKIs. Ceritinib and afatinib combination treatment partially restored the sensitivity to ceritinib. (C) 2016 AACR.

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Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

Makoto Nishino, Mari Ozaki, Ahmed E. Hegab, Junko Hamamoto, Shizuko Kagawa, Daisuke Arai, Hiroyuki Yasuda, Katsuhiko Naoki, Kenzo Soejima, Hideyuki Saya, Tomoko Betsuyaku

JOURNAL OF CANCER (IVYSPRING INT PUBL) 8 ( 10 ) 1774 - 1785 2017

ISSN 1837-9664

Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines.
Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS).
Results: Both CD44v(high)/ALDH(high) and CD44v(high)/ALDH(low) cells were enriched in cells with CSC characteristics, with the CD44v(high)/ALDH(low) cells being more proliferative and more resistant to chemotherapeutics, whereas CD44v(high)/ALDH(high) cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44v(high)/ALDH(low) cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44v(high)/ALDH(high) and CD44v(high)/ALDH(low) cells. CD44v(high) expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression.
Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44v(high)/ALDH(low) cells and others to CD44v(high)/ALDH(high) cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

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Activation of EGFR Bypass Signaling by TGF alpha Overexpression Induces Acquired Resistance to Alectinib in ALK-Translocated Lung Cancer Cells

Tetsuo Tani, Hiroyuki Yasuda, Junko Hamamoto, Aoi Kuroda, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Masayoshi Miyawaki, Ichiro Kawada, Katsuhiko Naoki, Yuichiro Hayashi, Tomoko Betsuyaku, Kenzo Soejima

MOLECULAR CANCER THERAPEUTICS (AMER ASSOC CANCER RESEARCH) 15 ( 1 ) 162 - 71 2016.01

ISSN 1535-7163

Alectinib is a highly selective ALK inhibitor and shows promising efficacy in non-small cell lung cancers (NSCLC) harboring the EML4-ALK gene rearrangement. The precise mechanism of acquired resistance to alectinib is not well defined. The purpose of this study was to clarify the mechanism of acquired resistance to alectinib in ALK-translocated lung cancer cells. We established alectinib-resistant cells (H3122-AR) from the H3122 NSCLC cell line, harboring the EML4-ALK gene rearrangement, by long-term exposure to alectinib. The mechanism of acquired resistance to alectinib in H3122-AR cells was evaluated by phospho-receptor tyrosine kinase (phospho-RTK) array screening and Western blotting. No mutation of the ALK-TK domain was found. Phospho-RTK array analysis revealed that the phosphorylation level of EGFR was increased in H3122-AR cells compared with H3122. Expression of TGF alpha, one of the EGFR ligands, was significantly increased and knockdown of TGF alpha restored the sensitivity to alectinib in H3122-AR cells. We found combination therapy targeting ALK and EGFR with alectinib and afatinib showed efficacy both in vitro and in a mouse xenograft model. We propose a preclinical rationale to use the combination therapy with alectinib and afatinib in NSCLC that acquired resistance to alectinib by the activation of EGFR bypass signaling. (C) 2015 AACR.

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In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer.

Hirano T, Yasuda H, Tani T, Hamamoto J, Oashi A, Ishioka K, Arai D, Nukaga S, Miyawaki M, Kawada I, Naoki K, Costa DB, Kobayashi SS, Betsuyaku T, Soejima K

Oncotarget (IMPACT JOURNALS LLC) 6 ( 36 ) 38789 - 803 2015.11

ISSN 1949-2553

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the "therapeutic window" of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.

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Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

Taicashi Sato, Kenzo Soejima, Eri Arai, Junko Hamamoto, Hiroyuki Yasuda, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Katsuhiko Naoki, Takashi Kohno, Koji Tsuta, Shun-Ichi Watanabe, Yae Kanai, Tomoko Betsuyaku

ONCOLOGY REPORTS (SPANDIDOS PUBL LTD) 34 ( 3 ) 1137 - 45 2015.09

ISSN 1021-335X

PTPRH is a receptor-type protein tyrosine phosphatase thought to be a potential regulator of tumorigenesis. The aim of the present study was to clarify the significance of PTPRH expression and its regulation by DNA methylation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma (LADC). PTPRH mRNA expression was examined in 89 NSCLC and corresponding non-cancerous tissues. The correlation between DNA methylation and PTPRH gene expression was investigated in another cohort that consisted of 145 patients with LADC, a major NSCLC subtype. Gene regulation by DNA methylation was assessed using a DNA methylation inhibitor. PTPRH mRNA expression was significantly upregulated in NSCLC. PTPRH DNA methylation was reduced in LADC samples and inversely correlated with mRNA expression. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines with low PTPRH expression, restored mRNA PTPRH expression levels. Furthermore, low PTPRH methylation was associated with shorter recurrence-free survival (P=1.64x10(-4)) and overall survival (P=5.54x10(-5)). Multivariate analysis revealed that PTPRH DNA methylation was an independent prognostic factor (P=6.88x10(-3)). It was confirmed that PTPRH is overexpressed in NSCLC. Furthermore, we determined that PTPRH is epigenetically regulated by DNA hypomethylation, with prognostic implications for LADC.

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Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma

Daisuke Arai, Ahmed E. Hegab, Kenzo Soejima, Aoi Kuroda, Kota Ishioka, Hiroyuki Yasuda, Katsuhiko Naoki, Shizuko Kagawa, Junko Hamamoto, Yongjun Yin, David M. Ornitz, Tomoko Betsuyaku

JOURNAL OF PATHOLOGY (WILEY-BLACKWELL) 235 ( 4 ) 593 - 605 2015.03

ISSN 0022-3417

Fibroblast growth factor 9 (FGF9) is essential for lung development and is highly expressed in a subset of human lung adenocarcinomas. We recently described a mouse model in which FGF9 expression in the lung epithelium caused proliferation of the airway epithelium at the terminal bronchioles and led to rapid development of adenocarcinoma. Here, we used this model to characterize the effects of prolonged FGF9 induction on the proximal and distal lung epithelia, and examined the propagation potential of FGF9-induced lung tumours. We showed that prolonged FGF9 over-expression in the lung resulted in the development of adenocarcinomas arising from both alveolar type II and airway secretory cells in the lung parenchyma and airways, respectively. We found that tumour cells harboured tumour-propagating cells that were able to form secondary tumours in recipient mice, regardless of FGF9 expression. However, the highest degree of tumour propagation was observed when unfractionated tumour cells were co-administered with autologous, tumour-associated mesenchymal cells. Although the initiation of lung adenocarcinomas was dependent on activation of the FGF9-FGF receptor 3 (FGFR3) signalling axis, maintenance and propagation of the tumour was independent of this signalling. Activation of an alternative FGF-FGFR axis and the interaction with tumour stromal cells is likely to be responsible for the development of this independence. This study demonstrates the complex role of FGF-FGFR signalling in the initiation, growth and propagation of lung cancer. Our findings suggest that analysing the expressions of FGF-FGFRs in human lung cancer will be a useful tool for guiding customized therapy. Copyright (c) 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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FOXD1 expression is associated with poor prognosis in non-small cell lung cancer.

Nakayama S, Soejima K, Yasuda H, Yoda S, Satomi R, Ikemura S, Terai H, Sato T, Yamaguchi N, Hamamoto J, Arai D, Ishioka K, Ohgino K, Naoki K, Betsuyaku T

Anticancer research (INT INST ANTICANCER RESEARCH) 35 ( 1 ) 261 - 8 2015.01

ISSN 0250-7005

Aim: Clinical microarray datasets were analyzed to search for new therapeutic targets and prognostic markers of non-small cell lung cancer (NSCLC). Materials and Methods: Microarray datasets from 90 lung cancer specimens, were analyzed with focus on the FOXD1 gene. Levels of FOXDI mRNA were assessed in lung cancer cell lines and these levels were correlated with survival. Results: FOXD1-knockdown led to suppression of cell proliferation. Moreover, patients with high FOXDI expression survived for a significantly shorter time than those with low FOXDI expression. Conclusion: The expression status of FOXD1 is a novel prognostic factor and may lead to new treatment strategies for NSCLC.

Multiple roles of extracellular fibroblast growth factors in lung cancer cells

Takahiro Suzuki, Hiroyuki Yasuda, Koji Funaishi, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Tetsuo Tani, Junko Hamamoto, Ayano Ohashi, Katsuhiko Naoki, Tomoko Betsuyaku, Kenzo Soejima

INTERNATIONAL JOURNAL OF ONCOLOGY (SPANDIDOS PUBL LTD) 46 ( 1 ) 423 - 9 2015.01

ISSN 1019-6439

Cancer cells are surrounded by the extracellular fluid, matrix, and stroma cells. Little is known about how extracellular components such as growth factor ligands affect the biology of lung cancer cells. The objective of this study was to determine whether extracellular fibroblast growth factors (FGFs) can affect the biology of lung cancer cells and to understand how extracellular FGFs affect the biology of lung cancer cells, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cells. Out of the 23 reported FGFs, we focused on FGF2, FGF9 and FGF10. We examined the effect of FGFs on proliferation, treatment sensitivity, and apoptosis of NSCLC (PC9) and SCLC (H69, H82 and H146) cells in vitro. To determine which FGF was the most clinically relevant, we also examined FGF2 and FGF9 concentrations in the serum of patients with lung cancer. We found that extracellular FGFs can affect proliferation, treatment sensitivity, and apoptosis of lung cancer cells in a cell-specific manner. Our results indicate that extracellular FGFs affect the biology of lung cancer cells through multiple functions.

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Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma

Junko Hamamoto, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Yuichiro Hayashi, Satoshi Yoda, Sohei Nakayama, Ryosuke Satomi, Hideki Terai, Shinnosuke Ikemura, Takashi Sato, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Tomoko Betsuyaku

CANCER SCIENCE (WILEY-BLACKWELL) 106 ( 1 ) 34 - 42 2015.01

ISSN 1347-9032

We identified transmembrane protease, serine 4 (TMPRSS4) as a putative, druggable target by screening surgically resected samples from 90 Japanese non-small-cell lung cancer (NSCLC) patients using cDNA microarray. TMPRSS4 has two druggable domains and was upregulated in 94.5% of the lung cancer specimens. Interestingly, we found that TMPRSS4 expression was associated with tissue factor pathway inhibitor 2 (TFPI-2) expression in these clinical samples. In contrast to TMPRSS4, TFPI-2 expression was downregulated in NSCLC samples. The in vitro induction of TFPI-2 in lung cancer cell lines decreased the expression of TMPRSS4 mRNA levels. Reporter assay showed that TFPI-2 inhibited transcription of TMPRSS4, although partially. Knockdown of TMPRSS4 reduced the proliferation rate in several lung cancer cell lines. When lung cancer cell lines were treated with 5-aza-2-deoxycytidine or trichostatin A, their proliferation rate and TMPRSS4 mRNA expression levels were also reduced through the upregulation of TFPI-2 by decreasing its methylation in vitro. The TFPI-2 methylation level in the low TMPRSS4 group appeared to be significantly low in NSCLC samples (P=0.02). We found a novel molecular mechanism that TFPI-2 negatively regulates cell growth by inhibiting transcription of TMPRSS4. We suggest that TMPRSS4 is upregulated by silencing of TFPI-2 through aberrant DNA methylation and contributes to oncogenesis in NSCLC.

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Long-term exposure to gefitinib induces acquired resistance through DNA methylation changes in the EGFR-mutant PC9 lung cancer cell line

Hideki Terai, Kenzo Soejima, Hiroyuki Yasuda, Takashi Sato, Katsuhiko Naoki, Shinnosuke Ikemura, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Junko Hamamoto, Yae Kanai, Tomoko Betsuyaku

INTERNATIONAL JOURNAL OF ONCOLOGY (SPANDIDOS PUBL LTD) 46 ( 1 ) 430 - 6 2015.01

ISSN 1019-6439

This study was designed to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). We established a gefitinib-resistant lung cancer cell line, PC9, which was originally gefitinib-sensitive, by serial long-term exposure to gefitinib. RNA and DNA were collected from both gefitinib-sensitive and -resistant PC9 cells, and comprehensive DNA methylation and mRNA expression analyses were performed using Infinium HumanMethylation27 Bead Arrays and Agilent SurePrint G3 Human Gene Expression 8x60K Array, respectively. DNA methylation was increased in 640 genes in gefitinib-resistant cells compared to parental cells. Among them, we selected 29 candidate genes that presented a decrease in mRNA expression in resistant PC9. We further studied four of the selected genes (C10orf116, IGFBP3, KL, and S100P) and found that KL or S100P silencing by siRNA induced a decrease in gefitinib sensitivity compared to that in the negative control in PC9. In conclusion, KL and S100P could be potential targets to overcome resistance to EGFR-TKIs.

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Claudin-1 is a novel target of miR-375 in non-small-cell lung cancer

Satoshi Yoda, Kenzo Soejima, Junko Hamamoto, Hiroyuki Yasuda, Sohei Nakayama, Ryosuke Satomi, Hideki Terai, Shinnosuke Ikemura, Takashi Sato, Katsuhiko Naoki, Tomoko Betsuyaku

LUNG CANCER (ELSEVIER IRELAND LTD) 85 ( 3 ) 366 - 72 2014.09

ISSN 0169-5002

Objectives: We previously reported low expression of miR-375 in squamous-cell carcinoma (SCC) and high expression in adenocarcinoma (AC) of the lung. miR-375's target genes and its function in non-small-cell lung cancer (NSCLC) have not been elucidated. Therefore, the present study was designed to identify the targets of miR-375 and to characterize its function in NSCLC.
Materials and methods: Candidate targets of miR-375 were determined using a prediction database and previous data on differential gene expression between SCC and AC. We evaluated miR-375 and target-gene expression levels in 12 NSCLC cell lines. The effect of miR-375 overexpression and knockdown was evaluated in NSCLC cell lines by transfecting them with an miR-375 precursor or inhibitor. A luciferase-reporter assay was performed to confirm a direct interaction between miR-375 and its target gene. Further, a wound-healing assay was performed to evaluate the effect of miR-375 overexpression on the migration of SK-MES-1 cells. Finally, to assess the clinical relevance, 63 clinical NSCLC samples were analyzed.
Results: Claudin-1 (CLDN1) has 4 putative miR-375 target sites in its 3'-untranslated region, and this gene was determined to be a target of miR-375. CLDN1 messenger RNA and protein expression were attenuated by overexpression of miR-375 and increased by knockdown of miR-375 in NSCLC cell lines. In a luciferase-reporter assay, miR-375 overexpression resulted in a 3-fold repression of luciferase activity (P<0.001). Cell migration was promoted by miR-375 overexpression, suggesting a high potential for invasion and metastasis in NSCLC expressing high levels of miR-375. In clinical NSCLC samples, there was a negative correlation between miR-375 and CLDN1 expression (r=-0.35, P=0.005). In addition, high miR-375 expression was correlated with a shorter survival time among the clinical samples (P=0.043).
Conclusion: CLDN1 is a novel target of miR-375, and high miR-375 expression shortens survival in NSCLC. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

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Expression of fibroblast growth factor 9 is associated with poor prognosis in patients with resected non-small cell lung cancer

Keiko Ohgino, Kenzo Soejima, Hiroyuki Yasuda, Yuichiro Hayashi, Junko Hamamoto, Katsuhiko Naoki, Daisuke Arai, Kota Ishioka, Takashi Sato, Hideki Terai, Shinnosuke Ikemura, Satoshi Yoda, Tetsuo Tani, Aoi Kuroda, Tomoko Betsuyaku

LUNG CANCER (ELSEVIER IRELAND LTD) 83 ( 1 ) 90 - 6 2014.01

ISSN 0169-5002

Objectives: Fibroblast growth factor (FGF) 9 is a member of the FGF family, which modulates cell proliferation, differentiation, and motility. Recent studies show that the activation of FGF signals including FGF9 is associated with the pathogenesis of several cancers; however, its clinicopathological and biological significance in non-small cell lung cancer (NSCLC) is unclear. The purpose of this study was to clarify the characteristics of NSCLC with FGF9 expression.
Materials and Methods: We evaluated the expression of FGF9 in resected NSCLC specimens and corresponding non-tumorous lung tissue samples using cDNA microarray and evaluated its clinicopathological characteristics.
Results: Nine out of 90 NSCLC specimens (10%) had "high"FGF9 expression compared with corresponding non-cancerous lung tissues. Histologically, of the 9 NSCLC specimens with high FGF9 expression, 5 were adenocarcinoma, whereas none were squamous cell carcinoma. FGF9 expression was not associated with sex, smoking history, or clinical stage. However, in patients with high and low FGF9 expression, the postoperative recurrence rates were 78% and 24% (p = 0.033), respectively. Overall survival was significantly shorter in patients with high FGF9 expression than in those with low FGF9 expression (p < 0.001).
Conclusion: Our data indicate that FGF9 may be a novel unfavorable prognostic indicator and a candidate therapeutic target of NSCLC. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

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Identification of microRNAs differentially expressed between lung squamous cell carcinoma and lung adenocarcinoma

Junko Hamamoto, Kenzo Soejima, Satoshi Yoda, Katsuhiko Naoki, Sohei Nakayama, Ryosuke Satomi, Hideki Terai, Shinnosuke Ikemura, Takashi Sato, Hiroyuki Yasuda, Yuichiro Hayashi, Michiie Sakamoto, Toru Takebayashi, Tomoko Betsuyaku

MOLECULAR MEDICINE REPORTS (SPANDIDOS PUBL LTD) 8 ( 2 ) 456 - 62 2013.08

ISSN 1791-2997

Recent advances in the treatment of non-small cell lung cancer (NSCLC) with new agents require accurate histological subtyping at diagnosis to avoid the higher risk of an adverse response and to obtain the maximum therapeutic response. However, interobserver variability, tumor hetero-geneity and the degree of differentiation may affect the decision concerning a pathological diagnosis of NSCLC. Therefore, the aim of this study was to identify specific microRNAs (miRNAs) as standardized biomarkers with high sensitivity and specificity in order to distinguish between squamous cell carcinoma (SCC) and adenocarcinoma (AC). Quantitative polymerase chain reaction (qPCR)-based miRNA array analysis was performed to identify microRNAs differentially expressed between SCC and AC using 86 resected NSCLC samples in addition to adjacent normal tissues. The results were confirmed by independent qRT-PCR assays with the same test samples and 88 additional validation samples, and from this we evaluated the usefulness of the identified miRNAs as biomarkers to distinguish between SCC and AC. Three miRNAs (hsa-miR-196b, hsa-miR-205 and hsa-miR-375) were identified. Discriminant analysis combining the three miRNAs appeared to distinguish SCC from AC accurately in the test and validation samples, demonstrating a sensitivity and specificity of 76 and 80%, and 85 and 83%, respectively. hsa-miR-196b, hsa-miR-205 and hsa-miR-375 were identified as biomarkers capable of distinguishing between lung SCC and lung AC. These newly identified miRNAs may prove to be highly valuable molecular markers for the classification of NSCLC histological subtypes and may contribute to the pathogenesis of each subtype of NSCLC.

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Activation of the FGF2-FGFR1 Autocrine Pathway: A Novel Mechanism of Acquired Resistance to Gefitinib in NSCLC

Hideki Terai, Kenzo Soejima, Hiroyuki Yasuda, Sohei Nakayama, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Keiko Ohgino, Shinnosuke Ikemura, Takashi Sato, Satoshi Yoda, Ryosuke Satomi, Katsuhiko Naoki, Tomoko Betsuyaku

MOLECULAR CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 11 ( 7 ) 759 - 67 2013.07

ISSN 1541-7786

Patients with non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations initially respond to EGFR-tyrosine kinase inhibitors (TKI) but eventually experience relapse. Acquired resistance to EGFR-TKIs is strongly associated with patient mortality. Thus, elucidation of the mechanism of acquired resistance to EGFR-TKIs is of great importance. In this study, gefitinib-resistant cell line models were established by long-term exposure to gefitinib using the gefitinib-sensitive lung cancer cell lines, PC9 and HCC827. Expression analyses indicated that both FGFR1 and FGF2 were increased in PC9 gefitinib-resistant (PC9 GR) cells as compared with PC9 naive (PC9 na) cells. Importantly, proliferation of gefitinib-resistant cells was dependent on the FGF2 -FGFR1 pathway. Mechanistically, inhibition of either FGF2 or FGFR1 by siRNA or FGFR inhibitor (PD173074) restored gefitinib sensitivity in PC9 GR cells. These data suggest that FGF2 (-)FGFR1 activation through an autocrine loop is a novel mechanism of acquired resistance to EGFR-TKIs.

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The PCR-invader method (structure-specific 5' nuclease-based method), a sensitive method for detecting EGFR gene mutations in lung cancer specimens; comparison with direct sequencing

Katsuhiko Naoki, Kenzo Soejima, Hiroaki Okamoto, Junko Hamamoto, Naoya Hida, Ichiro Nakachi, Hiroyuki Yasuda, Sohei Nakayama, Satoshi Yoda, Ryosuke Satomi, Shinnosuke Ikemura, Hideki Terai, Takashi Sato, Koshiro Watanabe

INTERNATIONAL JOURNAL OF CLINICAL ONCOLOGY (SPRINGER TOKYO) 16 ( 4 ) 335 - 44 2011.08

ISSN 1341-9625

Background Several sensitive assays, including the PCR-invader method (structure-specific 5' nuclease-based method), have been used to detect EGFR mutations in non-small-cell lung cancer (NSCLC). However, validation has not been reported. We assessed the detection rate of EGFR mutation by the PCR-invader method and direct sequencing using same clinical specimens.
Patients and methods EGFR mutations were analyzed with the PCR-invader method and compared with direct sequencing using paraffin tissues and pleural and pericardial effusions from NSCLC patients. The relationships between the treatment responses and mutations were evaluated retrospectively.
Results Fifty-four samples from 42 NSCLC patients were studied. EGFR mutations were identified in 52% of the patients and 52% of the samples with the PCR-invader method, but only in 43% of the patients and in 35% of the samples by direct sequencing. In the samples obtained from the same patients at different sites and different times, EGFR mutations were coincident in nine out of ten patients by the PCR-invader method but in six out of ten patients by direct sequencing. Seventeen patients with EGFR mutations were treated with gefitinib; the response rate (RR) and disease control rate (DCR) were 41 and 94%, and median treatment duration was more than 6 months. Seven EGFR mutation-negative patients were treated with gefitinib; the RR and DCR were 0 and 14%, and median treatment duration was 1 month.
Conclusion The PCR-invader method was useful for detecting EGFR mutations in clinical lung cancer specimens and is more sensitive than direct sequencing.

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Identification of a novel transmembrane domain involved in the negative modulation of mGluR1 using a newly discovered allosteric mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholin-4-ylethoxy)-4H-chromen-4-one

Fukuda, Junko, Suzuki, Gentaroh, Kimura, Toshifumi, Nagatomi, Yasushi, Ito, Satoru, Kawamoto, Hiroshi, Ozaki, Satoshi, Ohta, Hisashi

NEUROPHARMACOLOGY (PERGAMON-ELSEVIER SCIENCE LTD) 57 ( 4 ) 438 - 445 2009.09

Research paper (scientific journal), Joint Work, ISSN 0028-3908

Currently tested allosteric modulators for metabotropic glutamate receptor 1 (mGluR1) are known to regulate the activity of mGluR1 mainly through transmembrane (TM) domain 6 and/or 7. We identified a novel interaction site, N760 in TM5, which negatively regulates activation of mGluR1 with a newly discovered selective mGluR1 antagonist, 3-cyclohexyl-5-fluoro-6-methyl-7-(2-morpholinA-ylethoxy)-4H-chromen-4-one (CFMMC). CFMMC inhibited L-glutamate-induced intracellular Ca(2+) mobilization ([Ca(2+)](i)) in Chinese hamster ovary (CHO) cells expressing recombinant human mGluR1 a with IC(50) value of 50 nM, whereas it did not inhibit [Ca(2+)](i) in CHO cells expressing human mGluR5a [IC(50): >10 mu M). To identify the amino acid residues critical for antagonism of CFMMC, we constructed various point mutants of human mGluR1 and evaluated them in [Ca(2+)](i) assays. The inhibitory effects of CFMMC were significantly affected in point mutations of either I725 in TM4 or N760 in TM5, as well as mutations of W798, F801 and Y805 in TM6 or T815 in TM7. Further studies revealed that antagonistic activities of not only CFMMC but also other, structurally unrelated, mGluR1 antagonists such as 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-298198) and Compound 1 were reduced in N760 mutated mGluR1a. These results indicate that some mGluR1 allosteric antagonists require N760 in TM5 to demonstrate negative modulation of mGluR1 in addition to the reported amino acid residues in TM6 and TM7. (C) 2009 Elsevier Ltd. All rights reserved.

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Pharmacological characterization of a new, orally active and potent allosteric metabotropic glutamate receptor 1 antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide (FTIDC)

Suzuki, Gentaroh, Kimura, Toshifumi, Satow, Akio, Kaneko, Naoki, Fukuda, Junko, Hikichi, Hirohiko, Sakai, Naoko, Maehara, Shunsuke, Kawagoe-Takaki, Hiroko, Hata, Mikiko, Azuma, Tomoko, Ito, Satoru, Kawamoto, Hiroshi, Ohta, Hisashi

JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS) 321 ( 3 ) 1144 - 1153 2007.06

Research paper (scientific journal), Joint Work, ISSN 0022-3565

A highly potent and selective metabotropic glutamate receptor (mGluR) 1 antagonist, 4-[1-(2-fluoropyridin-3-yl)-5-methyl-1H-1,2, 3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)carboxamide( FTIDC), is described. FTIDC inhibits, with equal potency, L-glutamate-induced intracellular Ca2+ mobilization in Chinese hamster ovary cells expressing human, rat, or mouse mGluR1a. The IC50 value of FTIDC is 5.8 nM for human mGluR1a and 6200 nM for human mGluR5. The maximal response in agonist concentration-response curves was reduced in the presence of higher concentrations of FTIDC, suggesting the inhibition in a noncompetitive manner. FTIDC at 10 mu M showed no agonistic, antagonistic, or positive allosteric modulatory activity toward mGluR2, mGluR4, mGluR6, mGluR7, or mGluR8. FTIDC did not displace [H-3]L-quisqualate binding to human mGluR1a, indicating FTIDC is an allosteric antagonist. Studies using chimeric and mutant receptors of mGluR1 showed that transmembrane (TM) domains 4 to 7, especially Phe801 in TM6 and Thr815 in TM7, play pivotal roles in the antagonism of FTIDC. FTIDC inhibited the constitutive activity of mGluR1a, suggesting that FTIDC acts as an inverse agonist of mGluR1a. Intraperitoneally administered FTIDC inhibited face-washing behavior elicited by a group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine in mice at doses that did not produce motor impairment. Oral administration of FTIDC also inhibited the face-washing behavior. FTIDC is a highly potent and selective allosteric mGluR1 antagonist and a compound having oral activity without species differences in its antagonistic activity on recombinant human, mouse, and rat mGluR1. FTIDC could therefore be a valuable tool for elucidating the functions of mGluR1 not only in rodents but also in humans.

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