Researcher Profile and Settings

Affiliation

• Institute for Genetic Medicine　Molecular Pathogenesis

Job Title

• Professor

Degree

• Ph.D.（1994/03 Osaka University）

URL

http://kaken.nii.ac.jp/d/r/30270573.ja.html

J-Global ID

• 201301097897264646

Research Interests

• recombinant virus vector   Stem cells   DHODH   Dementia   Eva1   oligodendrocyte precursor cell   Ecrg4   senescence   neural stem/precursor cells   グリオーマ幹細胞   エピジェネティクス   Sox   Fas   細胞死   

Research Areas

• Life sciences / Tumor biology
• Life sciences / Neuroscience - general
• Life sciences / Medical biochemistry

Educational Organization

•  Master's degree program　Graduate School of Medicine
•  Doctoral (PhD) degree program　Graduate School of Medicine

Academic & Professional Experience

• 2023/04 - Today Hokkaido University Institute for Genetic Medicine Professor
• 2009 - 2012 Ehime University Professor
• 2005 - 2010 RIKEN Team leader
• 2002 - 2005 Cambridge University Group leader
• 2001 - 2002 Kumamoto University Associate professor
• 1998 - 2001 University College London Research fellow
• 1994 - 1998 Osaka Bioscience Institute Post doc

Education

• 1988 - 1990  Osaka university  Graduate school of medicine, master course
•        -   Osaka university  Graduate school of medicine, doctor course

Association Memberships

• 北海道癌談話会   THE JAPANESE ASSOCIATION FOR MOLECULAR TARGET THERAPY OF CANCER   THE JAPANESE CANCER ASSOCIATION   

Research Activities

Published Papers

• Combination of blockade of endothelin signalling and compensation of IGF1 expression protects the retina from degeneration

  Naoya Shigesada, Naoya Shikada, Manabu Shirai, Michinori Toriyama, Fumiaki Higashijima, Kazuhiro Kimura, Toru Kondo, Yasumasa Bessho, Takuma Shinozuka, Noriaki Sasai

  Cellular and Molecular Life Sciences 81 (1) 1420-682X 2024/01/22 

  Abstract Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, affecting 1 in 4000 people worldwide. This disease is currently assumed to be intractable, because effective therapeutic methods have not been established, regardless of genetic or sporadic traits. Here, we examined a RP mouse model in which the Prominin-1 (Prom1) gene was deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, conducted single-cell expression profiling, and compared the gene expression with and without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, Igf1 was decreased in rod photoreceptor cells and astrocytes under the light-stimulated condition. Consistently, the insulin-like growth factor (IGF) signal was weakened in light-stimulated photoreceptor cells. The recovery of Igf1 expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, thereby resulting in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in maintaining retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.
• Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition

  Haruka Wada, Ryo Otsuka, Wilfred T V Germeraad, Tomoki Murata, Toru Kondo, Ken-ichiro Seino

  Journal for ImmunoTherapy of Cancer 11 (11) e006677 - e006677 2023/11/13 

  Background The cancer stem cell theory proposes that tumor formation in vivo is driven only by specific tumor-initiating cells having stemness; however, clinical trials conducted to test drugs that target the tumor stemness provided unsatisfactory results thus far. Recent studies showed clear involvement of immunity in tumors; however, the requirements of tumor-initiation followed by stable growth in immunocompetent individuals remain largely unknown. Methods To clarify this, we used two similarly induced glioblastoma lines, 8B and 9G. They were both established by overexpression of an oncogenic H-RasL61 in p53-deficient neural stem cells. In immunocompromised animals in an orthotopic transplantation model using 1000 cells, both show tumor-forming potential. On the other hand, although in immunocompetent animals, 8B shows similar tumor-forming potential but that of 9G’s are very poor. This suggests that 8B cells are tumor-initiating cells in immunocompetent animals. Therefore, we hypothesized that the differences in the interaction properties of 8B and 9G with immune cells could be used to identify the factors responsible for its tumor forming potential in immunocompetent animals and performed analysis. Results Different from 9G, 8B cells induced senescence-like state of macrophages around tumors. We investigated the senescence-inducing factor of macrophages by 8B cells and found that it was interleukin 6. Such senescence-like macrophages produced Arginase-1, an immunosuppressive molecule known to contribute to T-cell hyporesponsiveness. The senescence-like macrophages highly expressed CD38, a nicotinamide adenine dinucleotide (NAD) glycohydrolase associated with NAD shortage in senescent cells. The addition of nicotinamide mononucleotide (NMN), an NAD precursor, in vitro inhibited to the induction of macrophage senescence-like phenotype and inhibited Arginase-1 expression resulting in retaining T-cell function. Moreover, exogenous in vivo administration of NMN after tumor inoculation inhibited tumor-initiation followed by stable growth in the immunocompetent mouse tumor model. Conclusions We identified one of the requirements for tumor-initiating cells in immunocompetent animals. In addition, we have shown that tumor growth can be inhibited by externally administered NMN against macrophage senescence-like state that occurs in the very early stages of tumor-initiating cell development. This therapy targeting the immunosuppressive environment formed by macrophage senescence-like state is expected to be a novel promising cancer therapeutic strategy.
• Selective vulnerability of human-induced pluripotent stem cells to dihydroorotate dehydrogenase inhibition during mesenchymal stem/stromal cell purification

  Ziadoon Al-Akashi, Denise Zujur, Daisuke Kamiya, Tomohisa Kato, Toru Kondo, Makoto Ikeya

  Frontiers in Cell and Developmental Biology 11 2023/02/06 [Refereed]
   

  The use of induced mesenchymal stem/stromal cells (iMSCs) derived from human induced pluripotent stem cells (hiPSCs) in regenerative medicine involves the risk of teratoma formation due to hiPSCs contamination in iMSCs. Therefore, eradicating the remaining undifferentiated hiPSCs is crucial for the effectiveness of the strategy. The present study demonstrates the Brequinar (BRQ)-induced inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, selectively induces apoptosis, cell cycle arrest, and differentiation; furthermore, it promotes transcriptional changes and prevents the growth of 3-dimensional hiPSC aggregates. Contrastingly, BRQ-treated iMSCs showed no changes in survival, differentiation potential, or gene expression. The results suggest that BRQ is a potential agent for the effective purification of iMSCs from a mixed population of iMSCs and hiPSCs, which is a crucial step in successful iMSC-based therapy.
• Glioblastoma-initiating cell heterogeneity generated by the cell-of-origin, genetic/epigenetic mutation and microenvironment.

  Kondo T

  Semin Cancer Biol. 2021 [Refereed][Invited]
  
• Tumor edge-to-core transition promotes malignancy in primary-to-recurrent glioblastoma progression in a PLAGL1/CD109-mediated mechanism

  Chaoxi Li, Hee Jin Cho, Daisuke Yamashita, Moaaz Abdelrashid, Qin Chen, Soniya Bastola, Gustavo Chagoya, Galal A Elsayed, Svetlana Komarova, Saya Ozaki, Yoshihiro Ohtsuka, Takeharu Kunieda, Harley I Kornblum, Toru Kondo, Do-Hyun Nam, Ichiro Nakano

  Neuro-Oncology Advances 2 (1) 2020/11/27 [Refereed][Not invited]
   

  Glioblastoma remains highly lethal due to its inevitable recurrence. Most of this recurrence is found locally, indicating that postsurgical tumor-initiating cells (TICs) accumulate at the tumor edge. These edge-TICs then generate local recurrence harboring new core lesions. Here, we investigated the clinical significance of the edge-to-core (E-to-C) signature generating glioblastoma recurrence and sought to identify its central mediators. First, we examined the association of E-to-C-related expression changes to patient outcome in matched primary and recurrent samples (n = 37). Specifically, we tested whether the combined decrease of the edge-TIC marker PROM1 (CD133) with the increase of the core-TIC marker CD109, representing E-to-C transition during the primary-to-recurrence progression, indicates poorer patient outcome. We then investigated the specific molecular mediators that trigger tumor recurrence driven by the E-to-C progression. Subsequently, the functional and translational significance of the identified molecule was validated with our patient-derived edge-TIC models in vitro and in vivo. Patients exhibiting the CD133low/CD109high signature upon recurrence representing E-to-C transition displayed a strong association with poorer progression-free survival and overall survival among all tested patients. Differential gene expression identified that PLAGL1 was tightly correlated with the core TIC marker CD109 and was linked to shorter patient survival. Experimentally, forced PLAGL1 overexpression enhanced, while its knockdown reduced, glioblastoma edge-derived tumor growth in vivo and subsequent mouse survival, suggesting its essential role in the E-to-C-mediated glioblastoma progression. E-to-C axis represents an ongoing lethal process in primary glioblastoma contributing to its recurrence, partly in a PLAGL1/CD109-mediated mechanism.
• Selective eradication of pluripotent stem cells by inhibiting DHODH activity

  Toru Kondo

  STEM CELLS 39 33 - 42 1066-5099 2020/10/12 [Refereed][Not invited]
  
• Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

  Smile Echizenya, Yukiko Ishii, Satoshi Kitazawa, Tadashi Tanaka, Shun Matsuda, Eriko Watanabe, Masao Umekawa, Shunsuke Terasaka, Kiyohiro Houkin, Tomohisa Hatta, Tohru Natsume, Yoshimasa Maeda, Shin-Ichi Watanabe, Shinji Hagiwara, Toru Kondo

  Neuro-oncology 22 (2) 229 - 239 2020/02/20 [Refereed][Not invited]
   

  BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1.  Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.
• Ecrg4 deficiency results in extended replicative capacity of neural stem cells in a Foxg1-dependent manner

  Yuka Nakatani, Hiroshi Kiyonari, Toru Kondo

  Development 146 (4) 0950-1991 2019/02/18 [Refereed][Not invited]
   

  The self-renewal activity of neural stem cells (NSCs) has been suggested to decrease with aging, resulting in age-dependent declines in brain function such as presbyopia and memory loss. The molecular mechanisms underlying decreases in NSC proliferation with age need to be elucidated in more detail to develop treatments that promote brain function. We previously reported that the expression of esophageal cancer-related gene 4 (Ecrg4) was up-regulated in aged NSCs, while its overexpression decreased NSC proliferation, suggesting a functional relationship between Ecrg4 and NSC aging. Using Ecrg4-deficient mice replaced the ecrg4 locus with the lacZ gene, we herein show that Ecrg4 deficiency recovered the age-dependent decline in NSC proliferation and enhanced spatial learning and memory in the Morris water-maze paradigm. We demonstrate that the proliferation of Ecrg4-deficient NSCs was partly maintained by the increased expression of Foxg1. Collectively, these results determine Ecrg4 as a NSC aging factor.
• Hedgehog Signal and Genetic Disorders.

  Noriaki Sasai, Michinori Toriyama, Toru Kondo

  Frontiers in genetics 10 1103 - 1103 2019 [Refereed][Not invited]
   

  The hedgehog (Hh) family comprises sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh), which are versatile signaling molecules involved in a wide spectrum of biological events including cell differentiation, proliferation, and survival; establishment of the vertebrate body plan; and aging. These molecules play critical roles from embryogenesis to adult stages; therefore, alterations such as abnormal expression or mutations of the genes involved and their downstream factors cause a variety of genetic disorders at different stages. The Hh family involves many signaling mediators and functions through complex mechanisms, and achieving a comprehensive understanding of the entire signaling system is challenging. This review discusses the signaling mediators of the Hh pathway and their functions at the cellular and organismal levels. We first focus on the roles of Hh signaling mediators in signal transduction at the cellular level and the networks formed by these factors. Then, we analyze the spatiotemporal pattern of expression of Hh pathway molecules in tissues and organs, and describe the phenotypes of mutant mice. Finally, we discuss the genetic disorders caused by malfunction of Hh signaling-related molecules in humans.
• Prominin-1 modulates Rho/ROCK-mediated membrane morphology and calcium-dependent intracellular chloride fulx.

  Hori, A., Nishide, K., Yasukuni, Y., Haga, K., Kakuta, W., Ishikawa, Y., Hayes, M.J., Ohnuma, S.I., Kiyonari, H., Kimura, K., Kondo, T., & Sasai, N.

  Sci. Rep. 9 15911  2019 [Refereed][Not invited]
  
• GPR17 is an essential regulator for the temporal adaptation of Sonic hedgehog signalling in neural tube development

  Atsuki Yatsuzuka, Akiko Hori, Minori Kadoya, Mami Matsuo-Takasaki, Toru Kondo, Noriaki Sasai

  Development 146 0950-1991 2019/01/01 [Refereed][Not invited]
   

  Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity in the neural progenitor cells changes dynamically during differentiation, but the mechanisms regulating this dynamicity are not fully understood. Here we show that temporal change of the intracellular cAMP level confers the temporal Shh signal, and the purinergic-type G-protein coupled receptor GPR17 plays an essential role for this regulation. GPR17 is highly expressed in the ventral progenitor regions of the neural tube and acts as a negative regulator of the Shh signal in chick embryos. While the activation of the GPR17-related signal inhibits ventral identity, perturbation of GPR17 expression leads to aberrant expansion of ventral neural domains. Notably, perturbation of GPR17 expression partially inhibits the negative feedback of Gli activity. Moreover, GPR17 increases cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulates Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.
• Ecrg4 peptide is the ligand of multiple scavenger receptors

  Tetsuo Moriguchi, Shuji Takeda, Shinzo Iwashita, Kei Enomoto, Tatsuya Sawamura, Uichi Koshimizu, Toru Kondo

  Scientific Reports 8 (1) 2018/03/06 [Refereed][Not invited]
   

  Abstract Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71–132 of Ecrg4 (Ecrg4(71–132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71–132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71–132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71–132) and transduce its signals, together with TLRs, in microglia.
• Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1

  Hiroki Kusumoto, Yoshihiko Hirohashi, Satoshi Nishizawa, Masamichi Yamashita, Kazuyo Yasuda, Aiko Murai, Akari Takaya, Takashi Mori, Terufumi Kubo, Munehide Nakatsugawa, Takayuki Kanaseki, Tomohide Tsukahara, Toru Kondo, Noriyuki Sato, Isao Hara, Toshihiko Torigoe

  Cancer Science 109 (3) 741 - 750 1349-7006 2018/03/01 [Refereed][Not invited]
   

  In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.
• Metabolomics-based discovery of a metabolite that enhances oligodendrocyte maturation

  Brittney A. Beyer, Mingliang Fang, Benjamin Sadrian, J. Rafael Montenegro-Burke, Warren C. Plaisted, Bernard P. C. Kok, Enrique Saez, Toru Kondo, Gary Siuzdak, Luke L. Lairson

  Nature Chemical Biology 14 (1) 22 - 28 1552-4469 2018/01/01 [Refereed][Not invited]
   

  Endogenous metabolites play essential roles in the regulation of cellular identity and activity. Here we have investigated the process of oligodendrocyte precursor cell (OPC) differentiation, a process that becomes limiting during progressive stages of demyelinating diseases, including multiple sclerosis, using mass-spectrometry-based metabolomics. Levels of taurine, an aminosulfonic acid possessing pleotropic biological activities and broad tissue distribution properties, were found to be significantly elevated (∼20-fold) during the course of oligodendrocyte differentiation and maturation. When added exogenously at physiologically relevant concentrations, taurine was found to dramatically enhance the processes of drug-induced in vitro OPC differentiation and maturation. Mechanism of action studies suggest that the oligodendrocyte-differentiation-enhancing activities of taurine are driven primarily by its ability to directly increase available serine pools, which serve as the initial building block required for the synthesis of the glycosphingolipid components of myelin that define the functional oligodendrocyte cell state.
• iPSC-based drug repositioning identifies Src/c-Abl as a therapeutic target for ALS motor neurons.

  Imamura K, Izumi Y, Watanabe A, Tsukita K, Woltjen K, Yamamoto T, Hotta A, Kondo T, Kitaoka S, Ohta A, Tanaka A, Watanabe D, Morita M, Kaji R, Takuma H, Tamaoka A, Kunath T, Wray S, Furuya H, Era T, Fujisawa T, Nishotoh H, Kengo H, Ichijo H, Julien J-P, Obata N, Hosokawa M, Akiyama H, Ayaki T, Ito H, Takahashi R, Yamanaka S, Inoue H

  Science Translational Medicine 9 391  2017/05 [Refereed][Not invited]
  
• Molecular mechanisms involved in gliomagenesis

  Toru Kondo

  BRAIN TUMOR PATHOLOGY 34 (1) 1 - 7 1433-7398 2017/01 [Refereed][Not invited]
   

  The application of molecular parameters in the World Health Organization classification of central nervous system tumors has advanced remarkably in this field. Large-scale genomic DNA analyses, including gene expression profiling, genome-wide association studies, and single-nucleotide polymorphism analysis, have revealed differences between tumors with the same pathological features. Because mutated genes and their signaling pathways can be targets for therapy, categorizing tumors by molecular parameters facilitates the selection of optimal therapeutic methods. Many genes are either oncogenes or tumor suppressor genes, and many of them are also involved in normal development, such as neural stem cell maintenance and neural differentiation. Moreover, genetic engineering has enabled the generation of tumors that phenocopy human tumors in mice. Here, I will discuss key molecular parameters, mechanisms of neural differentiation, isocitrate dehydrogenases, 1p36/19q13, and p53 in gliomagenesis. Because future therapeutic methods will be determined by the molecular mechanisms of tumors, identification of new parameters is still needed for further classification of glioma.
• Ecrg4 contributes to the anti-glioma immunosurveillance through type-I interferon signaling

  Tetsuo Moriguchi, Shun Kaneumi, Shuji Takeda, Kei Enomoto, Shyam Kumar Mishra, Tetsuro Miki, Uichi Koshimizu, Hidemitsu Kitamura, Toru Kondo

  OncoImmunology 5 (12) e1242547  2162-402X 2016/12/01 [Refereed][Not invited]
   

  Esophageal cancer-related gene 4 (Ecrg4), a hormone-like peptide, is thought to be a tumor suppressor, however, little is known about the mechanism of how Ecrg4 suppresses tumorigenesis. Here, we show that the ecrg4 null glioma-initiating cell (GIC) line, which was generated from neural stem cells of ecrg4 knockout (KO) mice, effectively formed tumors in the brains of immunocompetent mice, whereas the transplanted ecrg4 wild type-GIC line GIC(+/+) was frequently eliminated. This was caused by host immune system including adaptive T cell responses, since depletion of CD4+, CD8+, or NK cells by specific antibodies in vivo recovered tumorigenicity of GIC(+/+). We demonstrate that Ecrg4 fragments, amino acid residues 71–132 and 133–148, which are produced by the proteolitic cleavage, induced the expression of pro-inflammatory cytokines in microglia in vitro. Moreover, blockades of type-I interferon (IFN) signaling in vivo, either depleting IFN-α/β receptor 1 or using stat1 KO mice, abrogated the Ecrg4-dependent antitumor activity. Together, our findings indicate a major antitumor function of Ecrg4 in enhancing host immunity via type-I IFN signaling, and suggest its potential as a clinical candidate for cancer immunotherapy.
• Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models

  Takuhiro Shoji, Ryuta Saito, Masashi Chonan, Ichiyo Shibahara, Aya Sato, Masayuki Kanamori, Yukihiko Sonoda, Toru Kondo, Naoto Ishii, Teiji Tominaga

  NEURO-ONCOLOGY 18 (8) 1120 - 1128 1522-8517 2016/08 [Refereed][Not invited]
   

  Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas.
• Chemical Screening Identifies EUrd as a Novel Inhibitor Against Temozolomide-Resistant Glioblastoma-Initiating Cells

  Yoshihiro Tsukamoto, Naoki Ohtsu, Smile Echizenya, Satoko Otsuguro, Ryosuke Ogura, Manabu Natsumeda, Mizuho Isogawa, Hiroshi Aoki, Satoshi Ichikawa, Masahiro Sakaitani, Akira Matsuda, Katsumi Maenaka, Yukihiko Fujii, Toru Kondo

  STEM CELLS 34 (8) 2016 - 2025 1066-5099 2016/08 [Refereed][Not invited]
   

  Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multi-modal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5'-nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3.
• Plasticity of lung cancer stem-like cells is regulated by the transcription factor HOXA5 that is induced by oxidative stress

  Hiroshi Saijo, Yoshihiko Hirohashi, Toshihiko Torigoe, Ryota Horibe, Akari Takaya, Aiko Murai, Terufumi Kubo, Toshimitsu Kajiwara, Tsutomu Tanaka, Yosuke Shionoya, Eri Yamamoto, Reo Maruyama, Munehide Nakatsugawa, Takayuki Kanaseki, Tomohide Tsukahara, Yasuaki Tamura, Yasushi Sasaki, Takashi Tokino, Hiromu Suzuki, Toru Kondo, Hiroki Takahashi, Noriyuki Sato

  ONCOTARGET 7 (31) 50043 - 50056 1949-2553 2016/08 [Refereed][Not invited]
   

  Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.
• Olfactory Receptor Family 7 Subfamily C Member 1 Is a Novel Marker of Colon Cancer-Initiating Cells and Is a Potent Target of Immunotherapy

  Rena Morita, Yoshihiko Hirohashi, Toshihiko Torigoe, Satoko Ito-Inoda, Akari Takahashi, Tasuku Mariya, Hiroko Asanuma, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Terufumi Kubo, Goro Kutomi, Toru Mizuguchi, Takeshi Terui, Kunihiko Ishitani, Satoshi Hashino, Toru Kondo, Nozomi Minagawa, Norihiko Takahashi, Akinobu Taketomi, Satoru Todo, Masahiro Asaka, Noriyuki Sato

  CLINICAL CANCER RESEARCH 22 (13) 3298 - 3309 1078-0432 2016/07 [Refereed][Not invited]
   

  Purpose: Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy. Experimental Design: Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model. Results: OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model. Conclusions: OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. (C) 2016 AACR.
• Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480

  Akari Takaya, Yoshihiko Hirohashi, Aiko Murai, Rena Morita, Hiroshi Saijo, Eri Yamamoto, Terufumi Kubo, Munehide Nakatsugawa, Takayuki Kanaseki, Tomohide Tsukahara, Yasuaki Tamura, Ichiro Takemasa, Toru Kondo, Noriyuki Sato, Toshihiko Torigoe

  PLOS ONE 11 (7) e0158903  1932-6203 2016/07 [Refereed][Not invited]
   

  Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDH(high)) cells or cell surface marker-positive cells including CD44(+) cells and CD133(+) cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.
• A Powerful CRISPR/Cas9-Based Method for Targeted Transcriptional Activation

  Shota Katayama, Tetsuo Moriguchi, Naoki Ohtsu, Toru Kondo

  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION 55 (22) 6452 - 6456 1433-7851 2016/05 [Refereed][Not invited]
   

  Targeted transcriptional activation of endogenous genes is important for understanding physiological transcriptional networks, synthesizing genetic circuits, and inducing cellular phenotype changes. The CRISPR/Cas9 system has great potential to achieve this purpose, however, it has not yet been successfully used to efficiently activate endogenous genes and induce changes in cellular phenotype. A powerful method for transcriptional activation by using CRISPR/Cas9 was developed. Replacement of a methylated promoter with an unmethylated one by CRISPR/Cas9 was sufficient to activate the expression of the neural cell gene OLIG2 and the embryonic stem cell gene NANOG in HEK293T cells. Moreover, CRISPR/Cas9-based OLIG2 activation induced the embryonic carcinoma cell line NTERA-2 to express the neuronal marker beta III-tubulin.
• Targeting the glioblastoma-initiating cell-associated antigens.

  Toru Kondo

  Cancer Cell and Metastasis. 3 1 - 5 2016 [Refereed][Invited]
  
• Eva1 Maintains the Stem-like Character of Glioblastoma-Initiating Cells by Activating the Noncanonical NF-kappa B Signaling Pathway

  Naoki Ohtsu, Yuka Nakatani, Daisuke Yamashita, Shiro Ohue, Takanori Ohnishi, Toru Kondo

  CANCER RESEARCH 76 (1) 171 - 181 0008-5472 2016/01 [Refereed][Not invited]
   

  Glioblastoma (GBM)-initiating cells (GIC) are a tumorigenic subpopulation that are resistant to radio-and chemotherapies and are the source of disease recurrence. Therefore, the identification and characterization of GIC-specific factors is critical toward the generation of effective GBM therapeutics. In this study, we investigated the role of epithelial V-like antigen 1 (Eva1, also known as myelin protein zero-like 2) in stemness and GBM tumorigenesis. Eva1 was prominently expressed in GICs in vitro and in stem cell marker (Sox2, CD15, CD49f)-expressing cells derived from human GBM tissues. Eva1 knockdown in GICs reduced their self-renewal and tumor-forming capabilities, where-as Eva1 overexpression enhanced these properties. Eva1 deficiency was also associated with decreased expression of stemness related genes, indicating a requirement for Eva1 in maintaining GIC pluripotency. We further demonstrate that Eva1 induced GIC proliferation through the activation of the RelB-dependent noncanonical NF-kappa B pathway by recruiting TRAF2 to the cytoplasmic tail. Taken together, our findings highlight Eva1 as a novel regulator of GIC function and also provide new mechanistic insight into the role of noncanonical NF-kappa B activation in GIC, thus offering multiple potential therapeutic targets for preclinical investigation in GBM. (C) 2015 AACR.
• Ceacam1L Modulates STAT3 Signaling to Control the Proliferation of Glioblastoma-Initiating Cells

  Sadahiro Kaneko, Yuka Nakatani, Tatsuya Takezaki, Takuichiro Hide, Daisuke Yamashita, Naoki Ohtsu, Takanori Ohnishi, Shunsuke Terasaka, Kiyohiro Houkin, Toru Kondo

  CANCER RESEARCH 75 (19) 4224 - 4234 0008-5472 2015/10 [Refereed][Not invited]
   

  Glioblastoma-initiating cells (GIC) are a tumorigenic cell subpopulation resistant to radiotherapy and chemotherapy, and are a likely source of recurrence. However, the basis through which GICs are maintained has yet to be elucidated in detail. We herein demonstrated that the carcinoembryonic antigen-related cell adhesion molecule Ceacam1L acts as a crucial factor in GIC maintenance and tumorigenesis by activating c-Src/STAT3 signaling. Furthermore, we showed that monomers of the cytoplasmic domain of Ceacam1L bound to c-Src and STAT3 and induced their phosphorylation, whereas oligomerization of this domain ablated this function. Our results suggest that Ceacam1L-dependent adhesion between GIC and surrounding cells play an essential role in GIC maintenance and proliferation, as mediated by signals transmitted by monomeric forms of the Ceacam1L cytoplasmic domain. (C) 2015 AACR.
• miR340 Suppresses the Stem-like Cell Function of Glioma-Initiating Cells by Targeting Tissue Plasminogen Activator

  Daisuke Yamashita, Toru Kondo, Shiro Ohue, Hisaaki Takahashi, Madoka Ishikawa, Ryo Matoba, Satoshi Suehiro, Shohei Kohno, Hironobu Harada, Junya Tanaka, Takanori Ohnishi

  CANCER RESEARCH 75 (6) 1123 - 1133 0008-5472 2015/03 [Refereed][Not invited]
   

  Glioma-initiating cells (GIC) have stem-like cell properties thought to be sufficient for recurrence, progression, and drug resistance in glioblastomas. In the present study, we defined miRNA (miR)-340 as a differentially expressed miRNA in human GICs that inhibit GIC-mediated tumorigenesis. Furthermore, we defined tissue plasminogen activator (PLAT) as a critical direct target of miR340 for inhibition. Among miRNAs screened, we found that miR340 expression was decreased in all human GICs and in human glioblastoma tissues, compared with human neural stem cells and normal brain tissues. miR340 overexpression in GICs suppressed their proliferative, invasive, and migratory properties in vitro, triggering cell senescence in vitro and inhibiting GIC-induced tumorigenesis in mouse brains. shRNA-mediated silencing of PLAT in GICs phenocopied the effects of miR340 overexpression in vitro and in vivo, suggesting a potential role for tissue factor in stem-like cell function. Taken together, our results identified miR340 as a tumor suppressor that functions in GIC to enforce PLAT blockade and ablate their stem-like functions. (C) 2015 AACR.
• Genetic Background and Light-Dependent Progression of Photoreceptor Cell Degeneration in Prominin-1 Knockout Mice

  Margaret Dellett, Noriaki Sasai, Kenji Nishide, Silke Becker, Vasiliki Papadaki, G. Astrid Limb, Anthony T. Moore, Toru Kondo, Shin-ichi Ohnuma

  INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 56 (1) 164 - 176 0146-0404 2015/01 [Refereed][Not invited]
   

  PURPOSE. Mutations in the Prominin-1 (Prom1) gene are known to cause retinitis pigmentosa and Stargardt disease, both of which are associated with progressive photoreceptor cell death. There are no effective therapies for either disorder. The aim of this study was to investigate the mechanism of the retinal degeneration in Prom1-deficient mouse models. METHODS. We constructed Prom1 knockout mice with two distinct genetic backgrounds of C57BL/6 and C57BL/6xCBA/NSlc, and investigated the photoreceptor degeneration by means of histology and functional tests. In addition, we examined the effect of light on the Prom1(-/-) retina by rearing the mice in the normal light/dark cycle and completely dark conditions. Finally, we investigated if the retinoic-acid derivative Fenretinide slowed the pace of retinal degeneration in these mouse models. RESULTS. The Prom1(-/-)-knockout mice with both backgrounds developed photoreceptor degeneration after eye opening, but the CB57/BL6-background mice developed photoreceptor cell degeneration much faster than the C57BL/6xCBA/NSlc mice, demonstrating genetic background dependency. Interestingly, our histologic and functional examination showed that the photoreceptor cell degeneration of Prom1-knockout mice was light-dependent, and was almost completely inhibited when the mutant mice were kept in the dark. The Prom1-knockout retina showed strong downregulation of expression of the visual cycle components, Rdh12 and Abca4. Furthermore, administration of Fenretinide, which lowers the level of the toxic lipofuscin, slowed the degeneration of photoreceptor cells. CONCLUSIONS. These findings improve our understanding of the mechanism of cell death in Prominin-1-related disease and provide evidence that fenretinide may be worth studying in human disease.
• Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells

  Rena Morita, Satoshi Nishizawa, Toshihiko Torigoe, Akari Takahashi, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Alice Sokolovskaya, Vitaly Kochin, Toru Kondo, Satoshi Hashino, Masahiro Asaka, Isao Hara, Yoshihiko Hirohashi, Noriyuki Sato

  CANCER SCIENCE 105 (4) 389 - 395 1349-7006 2014/04 [Refereed][Not invited]
   

  The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy.
• Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells

  Rena Morita, Satoshi Nishizawa, Toshihiko Torigoe, Akari Takahashi, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Alice Sokolovskaya, Vitaly Kochin, Toru Kondo, Satoshi Hashino, Masahiro Asaka, Isao Hara, Yoshihiko Hirohashi, Noriyuki Sato

  Cancer Science 105 (4) 389 - 395 1349-7006 2014 [Refereed][Not invited]
   

  The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.
• Ectopically Expressed Variant Form of Sperm Mitochondria-Associated Cysteine-Rich Protein Augments Tumorigenicity of the Stem Cell Population of Lung Adenocarcinoma Cells

  Akari Takahashi, Yoshihiko Hirohashi, Toshihiko Torigoe, Yasuaki Tamura, Tomohide Tsukahara, Takayuki Kanaseki, Vitaly Kochin, Hiroshi Saijo, Terufumi Kubo, Munehide Nakatsugawa, Hiroko Asanuma, Tadashi Hasegawa, Toru Kondo, Noriyuki Sato

  PLOS ONE 8 (11) e69095  1932-6203 2013/11 [Refereed][Not invited]
   

  Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have self-renewal ability, differentiation ability and high tumor-initiating ability. CSCs/CICs are resistant to cancer therapies including chemotherapy and radiotherapy. Therefore, CSCs/CICs are thought to be responsible for cancer recurrence and distant metastasis after treatment. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we isolated CSCs/CICs as side population (SP) cells from lung carcinoma, colon carcinoma and breast carcinoma cells and analyzed the molecular mechanisms of CSCs/CICs. cDNA micro-array screening and RT-PCR analysis revealed that sperm mitochondria-associated cysteine-rich protein (SMCP) is ectopically expressed in SP cells. 5'-Rapid amplification of cDNA end (RACE) analysis revealed that the SMCP transcript in SP cells was a variant form (termed vt2) which is composed from only one exon. SMCP vt2 was detected in only cancer cells, whereas the wild-type (vt1) form of SMCP was expressed in the testis. SMCP was shown to have a role in tumor initiation by SMCP overexpression and SMCP knockdown using siRNAs in lung cancer cells. Taken together, the initiation results indicate that an ectopically expressed variant form of SMCP has a role in tumor initiation of CSCs/CICs and that the variant form of SMCP might be a novel CSC/CIC marker and a potential and promising target of CSC/CIC-targeting therapy.
• A regenerative approach to the treatment of multiple sclerosis

  Vishal A. Deshmukh, Virginie Tardif, Costas A. Lyssiotis, Chelsea C. Green, Bilal Kerman, Hyung Joon Kim, Krishnan Padmanabhan, Jonathan G. Swoboda, Insha Ahmad, Toru Kondo, Fred H. Gage, Argyrios N. Theofilopoulos, Brian R. Lawson, Peter G. Schultz, Luke L. Lairson

  NATURE 502 (7471) 327 - + 0028-0836 2013/10 [Refereed][Not invited]
   

  Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-inducedmodel of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.
• Preferential expression of cancer/testis genes in cancer stem-like cells: proposal of a novel sub-category, cancer/testis/stem gene

  R. Yamada, A. Takahashi, T. Torigoe, R. Morita, Y. Tamura, T. Tsukahara, T. Kanaseki, T. Kubo, K. Watarai, T. Kondo, Y. Hirohashi, N. Sato

  TISSUE ANTIGENS 81 (6) 428 - 434 0001-2815 2013/06 [Refereed][Not invited]
   

  Cancer/testis (CT) antigens encoded by CT genes are immunogenic antigens, and the expression of CT gene is strictly restricted to only the testis among mature organs. Therefore, CT antigens are promising candidates for cancer immunotherapy. In a previous study, we identified a novel CT antigen, DNAJB8. DNAJB8 was found to be preferentially expressed in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), and it is thus a novel CSC antigen. In this study, we hypothesized that CT genes are preferentially expressed in CSCs/CICs rather than in non-CSCs/-CICs and we examined the expression of CT genes in CSCs/CICs. The expression of 74 CT genes was evaluated in side population (SP) cells (=CSC) and main population (MP) cells (=non-CSC) derived from LHK2 lung adenocarcinoma cells, SW480 colon adenocarcinoma cells and MCF7 breast adenocarcinoma cells by RT-PCR and real-time PCR. Eighteen genes (MAGEA2, MAGEA3, MAGEA4, MAGEA6, MAGEA12, MAGEB2, GAGE1, GAGE8, SPANXA1, SPANXB1, SPANXC, XAGE2, SPA17, BORIS, PLU-1, SGY-1, TEX15 and CT45A1) showed higher expression levels in SP cells than in MP cells, whereas 10 genes (BAGE1, BAGE2, BAGE4, BAGE5, XAGE1, LIP1, D40, HCA661, TDRD1 and TPTE) showed similar expression levels in SP cells and MP cells. Thus, considerable numbers of CT genes showed preferential expression in CSCs/CICs. We therefore propose a novel sub-category of CT genes in this report: cancer/testis/stem (CTS) genes.
• DNA methyltransferase 1 is essential for initiation of the colon cancers

  Rena Morita, Yoshihiko Hirohashi, Hiromu Suzuki, Akari Takahashi, Yasuaki Tamura, Takayuki Kanaseki, Hiroko Asanuma, Satoko Inoda, Toru Kondo, Satoshi Hashino, Tadashi Hasegawa, Takashi Tokino, Minoru Toyota, Masahiro Asaka, Toshihiko Torigoe, Noriyuki Sato

  EXPERIMENTAL AND MOLECULAR PATHOLOGY 94 (2) 322 - 329 0014-4800 2013/04 [Refereed][Not invited]
   

  DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs. (C) 2012 Elsevier Inc. All rights reserved.
• Molecular markers of glioma initiating cells.

  Kondo, T

  Inflamation and Regeneration 33 181 - 189 2013 [Refereed][Not invited]
  
• HSP DNAJB8 Controls Tumor-Initiating Ability in Renal Cancer Stem-like Cells

  Satoshi Nishizawa, Yoshihiko Hirohashi, Toshihiko Torigoe, Akari Takahashi, Yasuaki Tamura, Takashi Mori, Takayuki Kanaseki, Kenjiro Kamiguchi, Hiroko Asanuma, Rena Morita, Alice Sokolovskaya, Junichi Matsuzaki, Ren Yamada, Reona Fujii, Harm H. Kampinga, Toru Kondo, Tadashi Hasegawa, Isao Hara, Noriyuki Sato

  CANCER RESEARCH 72 (11) 2844 - 2854 0008-5472 2012/06 [Refereed][Not invited]
   

  Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC. Cancer Res; 72(11); 2844-54. (C)2012 AACR.
• Efficiency of G2/M-related tumor-associated antigen-targeting cancer immunotherapy depends on antigen expression in the cancer stem-like population

  Takashi Mori, Satoshi Nishizawa, Yoshihiko Hirohashi, Toshihiko Torigoe, Yasuaki Tamura, Akari Takahashi, Vitaly Kochin, Reona Fujii, Toru Kondo, Mark I. Greene, Isao Hara, Noriyuki Sato

  EXPERIMENTAL AND MOLECULAR PATHOLOGY 92 (1) 27 - 32 0014-4800 2012/02 [Refereed][Not invited]
   

  The aim of this study was to establish a novel efficient cancer DNA vaccine approach. Many tumor-associated antigens (TAAs) have been reported; however, there is little information of the efficiency of each TAA. Normal cells barely undergo mitosis, whereas cancer cells divide frequently and grow well. Thus. G2/M-related antigens are cancer cell-specific and are regarded to be suitable candidates as targets of cancer immunotherapy. In this study, we compared the efficiencies of G2/M-related antigens including Birc5, Aurka, Nke2 and Plk1 by using a DNA vaccination model. Mice that had been immunized with G2/M-related antigens coding plasmid were challenged with CT26 colon cancer cells. Interestingly, Birc5- and Aurka-immunized mice showed an anti-tumor effect, whereas Nek2- and Plk1-immunized mice did not show any anti-tumor effect. We investigated the expression of G2/M-related antigens in cancer stem-like cell (CSC)/cancer-initiating cell (CIC) population to verify the difference in the anti-tumor effect. CSCs/CICs were isolated as side population (SP) cells using Hoechst 33342 dye from CT 26 cells. It was found that Birc5 and Aurka are expressed in both CSCs/CICs and non-CSCs/CICs (shared antigens), whereas Nek2 and Plk1 are expressed preferentially in non-CSCs/CICs (non-CSC antigens). Therefore, antigen expression in the CSC/CIC population might be related to the anti-tumor efficiency of cancer immunotherapy. Furthermore, we established a heat shock protein (Hsp90)-fused Birc5 plasmid to improve anti-cancer immunity. Birc5 fused to the N-terminal region of Hsp90 showed a stronger anti-tumor effect, whereas Birc5 fused to the C-terminal region of Hsp90 did not show enhancement compared with Birc5. These observations indicate that expression in the CSC/CIC population is essential to achieve tumor regression and that fusing antigens to the N-terminal region of Hsp90 enhances the anti-tumor effect. (C) 2011 Elsevier Inc. All rights reserved.
• SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity

  Munehide Nakatsugawa, Akari Takahashi, Yoshihiko Hirohashi, Toshihiko Torigoe, Satoko Inoda, Masaki Murase, Hiroko Asanuma, Yasuaki Tamura, Rena Morita, Yoshitaka Michifuri, Toru Kondo, Tadashi Hasegawa, Hiroki Takahashi, Noriyuki Sato

  LABORATORY INVESTIGATION 91 (12) 1796 - 1804 0023-6837 2011/12 [Refereed][Not invited]
   

  Recently, the SOX2 gene has been reported to be amplified in human lung squamous cell carcinomas. However, its roles in human lung adenocarcinomas are still elusive. In this study, we analyzed the functions of SOX2 in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) derived from human lung adenocarcinoma. Human lung CSCs/CICs were isolated as higher tumorigenic side population (SP) cells using Hoechst 33342 dye from several lung cancer cell lines. Four of nine lung cancer cell lines were positive for SP cells (LHK2, 1-87, A549, Lc817). The ratios of SP cells ranged from 0.4% for Lc817 to 2.8% for LHK2. To analyze the molecular aspects of SP cells, we performed microarray screening and RT-PCR analysis, and isolated SOX2 as one of a SP cell-specific gene. SOX2 was expressed predominantly in LHK2 and 1-87 SP cells, and was also expressed in several other cancer cell lines. The expression of SOX2 protein in primary human lung cancer tissues were also confirmed by immunohistochemical staining, and SOX2 was detected in more than 80% of primary lung cancer tissues. To address SOX2 molecular functions, we established a SOX2-overexpressed LHK2 and A549 cell line (LHK2-SOX2 and A549-SOX2). LHK2-SOX2 cells showed higher rates of SP cells and higher expression of POU5F1 compared with control cells. LHK2-SOX2 and A549-SOX2 cells showed relatively higher tumorigenicity than control cells. On the other hand, SOX2 mRNA knockdown of LHK2 SP cells by gene-specific siRNA completely abrogated tumorigenicity in vivo. These observations indicate that SOX2 has a role in maintenance of stemness and tumorigenicity of human lung adenocarcinoma CSCs/CICs and is a potential target for treatment. Laboratory Investigation (2011) 91, 1796-1804; doi:10.1038/labinvest.2011.140; published online 19 September 2011
• CD133(+) Cancer Stem Cell-like Cells Derived from Uterine Carcinosarcoma (Malignant Mixed Mullerian Tumor)

  Batsuren Choijamts, Shiro Jimi, Toru Kondo, Yasuko Naganuma, Taichi Matsumoto, Masahide Kuroki, Hiroshi Iwasaki, Makoto Emoto

  STEM CELLS 10 29 (10) 1485 - 1495 1066-5099 2011/10 [Refereed][Not invited]
   

  Cancer stem cells (CSCs) that display tumor-initiating properties have recently been identified. CD133, a surface glycoprotein linked to organ-specific stem cells, has been described as a marker of CSCs in different tumor types. We herein identify and characterize CSCs in human uterine carcinosarcoma (malignant mixed Mullerian tumor), which is one of the most aggressive and therapy-resistant gynecological malignancies and is considered to be of mesodermal origin. The CD133(+) population was increased in uterine carcinosarcoma, and this population showed biphasic properties in the primary tumor. CD133(+) cells predominantly formed spheres in culture and were able to differentiate into mesenchymal lineages. CD133(+) cells were more resistant to cisplatin/paclitaxel-induced cytotoxicity in comparison with CD133(-) cells. A real-time polymerase chain reaction analysis of the genes implicated in stem cell maintenance revealed that CD133(+) cells express significantly higher levels of Oct4, Nanog, Sox2, and Bmi1 than CD133(-) cells. Moreover, CD133(+) cells showed a high expression level of Pax2 and Wnt4, which are genes essential for Mullerian duct formation. These CD133(+) cells form serially transplantable tumors in vivo and the resulting CD133(+) tumors replicated the EpCAM, vimentin, and estrogen and progesterone receptor expression of the parent tumor, indicating that CSCs likely differentiated into cells comprising the uterine carcinosarcoma tissue. Moreover, strong CD133 expression in both epithelial and mesenchymal elements in primary tumor demonstrated significant prognostic value. These findings suggest that CD133(+) cells have the characteristics of CSCs and Mullerian mesenchymal progenitors. STEM CELLS 2011;29:1485-1495
• Essential role of the Hedgehog signaling pathway in human glioma-initiating cells

  Tatsuya Takezaki, Takuichiro Hide, Hiromi Takanaga, Hideo Nakamura, Jun-ichi Kuratsu, Toru Kondo

  CANCER SCIENCE 102 (7) 1306 - 1312 1347-9032 2011/07 [Refereed][Not invited]
   

  Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme, contain cancer-initiating cells (also known as cancer stem cells), which self-renew and are malignant, with features of tissue-specific stem cells. As these cells are resistant to irradiation and anti-cancer drugs, it is important to characterize them and find targeting therapies. In this study, we established two primary human glioma cell lines from anaplastic oligodendroglioma and glioblastoma multiforme. These lines were enriched in glioma-initiating cells, as just 10 cells formed malignant glioma when injected into mouse brain. We used these cell lines to examine the roles of the Notch, Hedgehog and Wnt signaling pathways, which are involved in stem-cell maintenance and tumorigenesis, to determine which of these pathways are crucial to glioma-initiating cells and their regulation. Here we show that the Hedgehog pathway is indispensable for glioma-initiating cell proliferation and tumorigenesis; the Hedgehog signaling inhibitors prevented glioma-initiating cell proliferation, while signaling inhibitors for Notch or Wnt did not. Overexpression of Gli2 Delta C, a C-terminal-truncated form of Gli2 that antagonizes Gli transcription factor functions, blocked glioma-initiating cell proliferation in culture and tumorigenesis in vivo. Knockdown of the Gli down-stream factor Cdc2 also prevented glioma-initiating cell proliferation. Taken together, these results show that the Hedgehog -> Gli -> Cdc2 signaling cascade plays a role in the proliferation and malignancy of glioma-initiating cells. (Cancer Sci 2011; 102: 1306-1312)
• Combination of a Ptgs2 inhibitor and an epidermal growth factor receptor-signaling inhibitor prevents tumorigenesis of oligodendrocyte lineage-derived glioma-initiating cells

  Takuichiro Hide, Tatsuya Takezaki, Yuka Nakatani, Hideo Nakamura, Jun-Ichi Kuratsu, Toru Kondo

  Stem Cells 29 (4) 590 - 599 1066-5099 2011/04 [Refereed][Not invited]
   

  Recent findings have demonstrated that malignant tumors, including glioblastoma multiforme (GBM), contain cancerinitiating cells (CICs also known as cancer stem cells), which self-renew and are malignant. However, it remains controversial whether such CICs arise from tissue-specific stem cells, committed precursor cells, or differentiated cells. Here, we sought to examine the origin of the CICs in GBM. We first showed that the overexpression of oncogenic HRasL61 transformed p53-deficient oligodendrocyte precursor cells (OPCs) and neural stem cells (NSCs) into glioma-initiating cell (GIC)-like cells in mice. When as few as 10 of these GIC-like cells were transplanted in vivo, they formed a transplantable GBM with features of human GBM, suggesting that these GIC-like cells were enriched in CICs. DNA microarray analysis showed that widespread genetic reprogramming occurred during the OPCs' transformation: they largely lost their OPC characteristics and acquired NSC ones, including the expression of prominin1, hmga2, ptgs2, and epiregulin. In addition, the combination of a Ptgs2 inhibitor and an epidermal growth factor receptor (EGFR)-signaling inhibitor prevented the tumorigenesis of transformed OPCs and human GICs (hGICs) obtained from anaplastic oligodendroglioma, but not of transformed NSCs or hGICs obtained from GBM. Together, these findings suggest that GBM can arise from either OPCs or NSCs and that the therapeutic targets for GBM might be different, depending on each GIC's cell-of-origin. © AlphaMed Press.
• Cytotoxic T Lymphocytes Efficiently Recognize Human Colon Cancer Stem-Like Cells

  Satoko Inoda, Yoshihiko Hirohashi, Toshihiko Torigoe, Rena Morita, Akari Takahashi, Hiroko Asanuma, Munehide Nakatsugawa, Satoshi Nishizawa, Yasuaki Tamura, Tetsuhiro Tsuruma, Takeshi Terui, Toru Kondo, Kunihiko Ishitani, Tadashi Hasegawa, Koichi Hirata, Noriyuki Sato

  AMERICAN JOURNAL OF PATHOLOGY 178 (4) 1805 - 1813 0002-9440 2011/04 [Refereed][Not invited]
   

  Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide. To evaluate the susceptibility of SP cells to CTIs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs. (Am J Pathol 2011, 178:1805-1813; DOI: 10.1016/j.ajpath.2011.01.004)
• Tumorigenesis of glioma-initiating cells: Role of Sox11.

  Toru Kondo

  Stem Cells and Cancer Stem Cells 1 93 - 98 2011 [Refereed][Invited]
  
• The Transcriptional Mediator Subunit MED1/TRAP220 in Stromal Cells Is Involved in Hematopoietic Stem/Progenitor Cell Support through Osteopontin Expression

  Akiko Sumitomo, Ruri Ishino, Norinaga Urahama, Kana Inoue, Kenji Yonezawa, Natsumi Hasegawa, Osamu Horie, Hiroshi Matsuoka, Toru Kondo, Robert G. Roeder, Mitsuhiro Ito

  MOLECULAR AND CELLULAR BIOLOGY 30 (20) 4818 - 4827 0270-7306 2010/10 [Refereed][Not invited]
   

  MED1/TRAP220, a subunit of the transcriptional Mediator/TRAP complex, is crucial for various biological events through its interaction with distinct activators, such as nuclear receptors and GATA family activators. In hematopoiesis, MED1 plays a pivotal role in optimal nuclear receptor-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we present evidence that MED1 in stromal cells is involved in supporting hematopoietic stem and/or progenitor cells (HSPCs) through osteopontin (OPN) expression. We found that the proliferation of bone marrow (BM) cells cocultured with MED1 knockout (Med1(-/-)) mouse embryonic fibroblasts (MEFs) was significantly suppressed compared to the control. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) was attenuated for BM cells cocultured with Med1(-/-) MEFs. The vitamin D receptor (VDR)- and Runx2-mediated expression of OPN, as well as Mediator recruitment to the Opn promoter, was specifically attenuated in the Med1(-/-) MEFs. Addition of OPN to these MEFs restored the growth of cocultured BM cells and the number of LTC-ICs, both of which were attenuated by the addition of the anti-OPN antibody to Med1(+/+) MEFs and to BM stromal cells. Consequently, MED1 in niche appears to play an important role in supporting HSPCs by upregulating VDR- and Runx2-mediated transcription on the Opn promoter.
• Mouse Induced Glioma-Initiating Cell Models and Therapeutic Targets

  Toru Kondo

  ANTI-CANCER AGENTS IN MEDICINAL CHEMISTRY 10 (6) 471 - 480 1871-5206 2010/07 [Refereed][Invited]
   

  Both stem cells and cancer cells are thought to be capable of unlimited self-renewal. Moreover, a small number of cancer cells express stem cell markers, including CD133 and ATP-binding cassette transporters through which the cells can pump out anti-cancer drugs or specific fluorescence dyes such as Hoechst33342, suggesting that either cancer cells resemble stem cells or that cancers contain stem cell-like cancer cells, called cancer-initiating cells (CICs) or cancer stem cells. Using the common characteristics of tissue-specific stem cells, malignant tumors and cancer cell lines were shown to contain CICs, which self-renew and are tumorigenic. CICs are also resistant to both irradiation and chemotherapy. These findings suggest that CICs are critical targets for successful therapy. However, CICs have not been well characterized, due to a lack of specific markers. We recently established mouse glioma-initiating cell (GIC) lines by overexpressing oncogenic HRas(L61) in p53-deficient neural cells. These cells form transplantable glioblastoma multiforme (GBM) with features of human GBM when as few as 10 cells are transplanted in vivo, suggesting that these GIC-like cells are enriched in CICs. Characterization of these GICs showed that they expressed little or no Sox11. The overexpression of exogenous Sox11 in GICs blocked their tumorigenesis by inducing their neuronal differentiation, which was accompanied by decreased levels of a novel oncogene, plagl1. These findings suggest that Sox11 and Plagl1 work as a tumor suppressor and oncogene, respectively, in GBM and are potential therapeutic targets.
• [Cancer stem cells and molecular targeting medicine].

  Kondo T

  Nihon rinsho. Japanese journal of clinical medicine 6 68 (6) 986 - 989 0047-1852 2010/06 [Refereed][Not invited]
  
• Esophageal cancer-related gene 4 is a secreted inducer of cell senescence expressed by aged CNS precursor cells

  Yuki Kujuro, Norihiro Suzuki, Toru Kondo

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 (18) 8259 - 8264 0027-8424 2010/05 [Refereed][Not invited]
   

  Mammalian aging is thought to be partially caused by the diminished capacity of stem/precursor cells to undergo self-renewing divisions. Although many cell-cycle regulators are involved in this process, it is unknown to what extent cell senescence, first identified as irreversible growth arrest in vitro, contributes to the aging process. Here, using a serum-induced mouse oligodendrocyte precursor cell (mOPC) senescence model, we identified esophageal cancer-related gene 4 (Ecrg4) as a senescence inducer with implications for the senescence-like state of postmitotic cells in the aging brain. Although mOPCs could proliferate indefinitely when cultured using the appropriate medium (OPC medium), they became senescent in the presence of serum and maintained their senescent phenotype even when the serum was subsequently replaced by OPC medium. We show that Ecrg4 was up-regulated in the senescent OPCs, its overexpression in OPCs induced senescence by accelerating the proteasome-dependent degradation of cyclins D1 and D3, and that its knockdown by a specific short hairpin RNA prevented these phenotypes. We also show that senescent OPCs secreted Ecrg4 and that recombinant Ecrg4 induced OPC senescence in culture. Moreover, increased Ecrg4 expression was observed in the OPCs and neural precursor cells in the aged mouse brain; this was accompanied by the expression of senescence-associated beta-galactosidase activity, indicating the cells' entrance into senescence. These results suggest that Ecrg4 is a factor linking neural-cell senescence and aging.
• Sox11 prevents tumorigenesis of glioma-initiating cells by inducing neuronal differentiation

  Takuichiro Hide, Tatsuya Takezaki, Yuka Nakatani, Hideo Nakamura, Jun-Ichi Kuratsu, Toru Kondo

  Cancer Research 69 (20) 7953 - 7959 0008-5472 2009/10/15 [Refereed][Not invited]
   

  Recent findings have shown that malignant tumors contain cancer-initiating cells (CIC), which self-renew and are tumorigenic. However, CICs have not been characterized properly due to lackof specific markers. We recently established a mouse glioma cell line, NSCL61, by overexpressing an oncogenic HRas L61 in p53-deficient neural stem cells. Using limiting dilution assays, we show that only 2 of 24 NSCL61 clones retained their tumorigenicity in vivo, although the others also expressed oncogenic HRasL61 and could proliferate in culture. A comparison of the gene expression profiles of tumorigenic and nontumorigenic clones showed that the tumorigenic clones had lost Sox11 expression. We show that overexpression of sox11 prevented tumorigenesis of NSCL61s by inducing their neuronal differentiation accompanied with decreased levels of plagl1. We also show that overexpression of plagl1 abolished neuronal commitment of nontumorigenic cells and induced them to become tumorigenic. Moreover, we show that human glioma-initiating cells lost sox11 expression, and overexpression of sox11 prevented their tumorigenesis in vivo. Together with the clinical evidence showing that downregulation of sox11 mRNA correlates with a significant decrease in survival, these findings suggest that Sox11 prevents gliomagenesis by blocking the expression of oncogenic plagl1. ©2009 American Association for Cancer Research.
• Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas

  M. Murase, M. Kano, T. Tsukahara, A. Takahashi, T. Torigoe, S. Kawaguchi, S. Kimura, T. Wada, Y. Uchihashi, T. Kondo, T. Yamashita, N. Sato

  BRITISH JOURNAL OF CANCER 101 (8) 1425 - 1432 0007-0920 2009/10 [Refereed][Not invited]
   

  BACKGROUND: Several human cancers have been found to contain cancer stem-like cells (CSCs) having cancer-initiating ability. However, only a few reports have shown the existence of CSCs in bone and soft tissue sarcomas. In this study, we identified and characterised side population (SP) cells that showed drug-resistant features in human bone sarcoma cell lines. METHODS: In seven osteosarcoma cell lines (OS2000, KIKU, NY, Huo9, HOS, U2OS and Saos2) and in one bone malignant fibrous histiocytoma (MFH) cell line (MFH2003), the frequency of SP cells was analysed. Tumourigenicity of SP cells was assessed in vitro and in vivo. Gene profiles of SP cells and other populations (main population; MP) of cells were characterised using cDNA microarrays. RESULTS: SP cells were found in NY (0.31%) and MFH2003 (5.28%). SP cells of MFH2003 formed spherical colonies and re-populated into SP and MP cells. In an NOD/SCID mice xenograft model, 1 x 10(3) sorted SP cell-induced tumourigenesis. cDNA microarray analysis showed that 23 genes were upregulated in SP cells. CONCLUSIONS: We showed that SP cells existed in bone sarcoma cell lines. SP cells of MFH2003 had cancer-initiating ability in vitro and in vivo. The gene profiles of SP cells could serve as candidate markers for CSCs in bone sarcomas. British Journal of Cancer (2009) 101, 1425-1432. doi: 10.1038/sj.bjc.6605330 www.bjcancer.com Published online 29 September 2009 (C) 2009 Cancer Research UK
• Glioblastoma Formation from Cell Population Depleted of Prominin1-Expressing Cells

  Kenji Nishide, Yuka Nakatani, Hiroshi Kiyonari, Toru Kondo

  PLOS ONE 4 (8) e6869  1932-6203 2009/08 [Refereed][Not invited]
   

  Prominin1 (Prom1, also known as CD133 in human) has been widely used as a marker for cancer stem cells (CSCs), which self-renew and are tumorigenic, in malignant tumors including glioblastoma multiforme (GBM). However, there is other evidence showing that Prom1-negative cancer cells also form tumors in vivo. Thus it remains controversial whether Prom1 is a bona fide marker for CSCs. To verify if Prom1-expressing cells are essential for tumorigenesis, we established a mouse line, whose Prom1-expressing cells can be eliminated conditionally by a Cre-inducible DTA gene on the Prom1 locus together with a tamoxifen-inducible CreER (TM), and generated glioma-initiating cells (GICs-LD) by overexpressing both the SV40 Large T antigen and an oncogenic H-Ras(L61) in neural stem cells of the mouse line. We show here that the tamoxifen-treated GICs-LD (GICs-DTA) form tumor-spheres in culture and transplantable GBM in vivo. Thus, our studies demonstrate that Prom1-expressing cells are dispensable for gliomagenesis in this mouse model.
• Common mechanism underlying oligodendrocyte development and oligodendrogliomagenesis

  Toru Kondo

  Brain and Nerve 61 (7) 741 - 751 1881-6096 2009/07 [Refereed][Invited]
   

  A number of extrinsic factors and intracellular mechanisms have been revealed to be involved in oligodendrocyte development. For instance, sonic hedgehog induces the expression of basic helix-loop-helix (bHLH) transcription factors, Olig1 and Olig2, and a homeobox transcription factor Nkx2.2, both of which induce neural stem cells (NSCs) to differentiate into oligodendrocyte precursor cells when the factors work together. In contrast, Notch and bone morphogenic proteins (BMP) block oligodendrocyte differentiation by inducing the expression of the transcription inhibitors Hes5 and Id4, respectively. Moreover, it was shown that other extrinsic and intrinsic factors, including platelet derived growth factor, thyroid hormone (TH), TH receptors, p53, and Wnt, are also involved in the development, positively or negatively. It has been thought that oligodendroglioma, one of brain tumors, is generated from oligodendrocyte lineage cells as the tumor cells share characteristics of oligodendrocyte, such as the honeycomb structure, and the expression of oligodendrocyte-related factors, including Olig2 and NG2 proteoglycan. However, recent progress in the field revealed that oligodendroglioma might be generated from NSCs and astrocytes as well as oligodendrocyte lineage cells. Therefore, it is crucial to investigate the cell-of-origin of oligodendroglioma and to identify target genes and markers for the therapy. In this review, I summarize the mechanism of oligodendrocyte development and present how the oligodendrocyte research can help to understand and to investigate the mechanism of oligodendroglioma development.
• Gli2 Is a Novel Regulator of Sox2 Expression in Telencephalic Neuroepithelial Cells

  Hiromi Takanaga, Nobuko Tsuchida-Straeten, Kenji Nishide, Akira Watanabe, Hiroyuki Aburatani, Toru Kondo

  STEM CELLS 27 (1) 165 - 174 1066-5099 2009 [Refereed][Not invited]
   

  Multipotential neural stem cells (NSCs) in the central nervous system (CNS) proliferate indefinitely and give rise to neurons, astrocytes, and oligodendrocytes. As NSCs hold promise for CNS regeneration, it is important to understand how their proliferation and differentiation are controlled. We show here that the expression of sox2 gene, which is essential for the maintenance of NSCs, is regulated by the Gli2 transcription factor, a downstream mediator of sonic hedgehog (Shh) signaling: Gli2 binds to an enhancer that is vital for sox2 expression in telencephalic neuroepithelial (NE) cells, which consist of NSCs and neural precursor cells. Overexpression of a truncated form of Gli2 (Gli2 triangle C) or Gli2-specific short hairpin RNA (Gli2 shRNA) in NE cells in vivo and in vitro inhibits cell proliferation and the expression of Sox2 and other NSC markers, including Hes1, Hes5, Notch1, CD133, and Bmi1. It also induces premature neuronal differentiation in the developing NE cells. In addition, we show evidence that Sox2 expression decreases significantly in the developing neuroepithelium of Gli2-deficient mice. Finally, we demonstrate that coexpression of Gli2 triangle C and Sox2 can rescue the expression of Hes5 and prevent premature neuronal differentiation in NE cells but cannot rescue its proliferation. Thus these data reveal a novel transcriptional cascade, involving Gli2 -> Sox2 -> Hes5, which maintains the undifferentiated state of telencephalic NE cells. STEM CELLS 2009; 27: 165- 174
• Brain tumor stem cells as research and treatment targets

  Takuichiro Hide, Tatsuya Takezaki, Hideo Nakamura, Junichi Kuratsu, Toru Kondo

  BRAIN TUMOR PATHOLOGY 25 (2) 67 - 72 1433-7398 2008/11 [Refereed][Not invited]
   

  Glioblastoma multiforme (GBM) is one of the most malignant forms of human cancer. Despite intensive treatment, the mean survival of GBM patients remains about 1 year. Recent cancer studies revealed that cancer tissues are pathologically heterogeneous and only a small population of cells has the specific ability to reinitiate cancer. This small cell population is called cancer stem cells (CSCs); in brain tumors these are known as brain tumor stem cells (BTSCs). The identification of BTSCs yielded new insights into chemo-and radioresistance, by which BTSCs can survive selectively and initiate recurrence. Research focused on BTSCs as treatment targets may contribute to the discovery of new therapeutic strategies.
• Making of glioma-initiating cells.

  Kondo, T

  Inflamation and Regeneration The Japanese Society of Inflammation and Regeneration 28 (6) 537 - 542 1880-9693 2008 [Refereed][Invited]
   

  There is an increasing body of evidence that suggests that malignant tumors, such as leukemia, breast cancers, and brain cancers, contain cells that maintain the characteristics of tissue-specific stem cells (TSSCs) and are malignant. Malignant gliomas, for example, contain proliferating cells expressing neural stem cell (NSC) markers, such as nestin and CD133, and differentiating cells expressing either neuronal markers or glial markers, raising the possibility that the tumors may contain NSC-like cancer cells. Additional evidence also exists, which indicate that malignant tumors might contain stem cell-like cancer cells, called "cancer stem cells" (CSCs) or "tumor initiating cells". Although a number of anti-cancer drugs and irradiation therapy have been successful in eliminating cancers, occasionally some cancer cells survive and invade other tissues, leading to the recurrence of cancer; this indicates that the surviving cells are not only resistant to such anti-cancer drugs and irradiation but are also malignant. Previous studies have shown that various ATP binding cassette transporters, such as the multi-drug resistant protein encoded by the multi-drug resistant gene, and the breast cancer resistant protein 1 (BCRP1), contribute to drug resistance in cancers. Interestingly, some of these transporters are also expressed in many types of normal stem cells. BCRP1, for example, excludes the fluorescent dye Hoechst 33342, identifying a side population (SP) enriched with TSSCs. Taking advantage of the common characteristics that exist between TSSCs and cancer cells, many groups have demonstrated that CSCs in tumors or cancer cell lines can self-renew, express well-known TSSC markers, form tumors when transplanted in vivo and show resistance to anti-cancer treatments; this suggests that CSCs are important targets for curable therapy. In order to develop an effective therapy against tumors, characterizing and finding ways to kill CSCs is essential. In this review, I have summarized the recent progresses made in glioma CSC research and discuss the perspectives of this field.
• Stem cell-like brain cancer cells

  Toru Kondo

  Current Cancer Therapy Reviews 4 (3) 201 - 205 1573-3947 2008 [Refereed][Invited]
   

  Both stem cells and cancer cells can proliferate indefinitely. In many case, cancers consist of the cells expressing tissue-specific stem cell markers and the cells expressing differentiation markers. Moreover, it has been revealed that many cancer cells express ATP-binding cassette (ABC) transporters, by which the cells pump out a specific fluorescence dyes as well as anti-cancer drugs. Thus these finding suggest that either cancer cells resemble stem cells or cancers contain stem cell-like cells. Using the common characteristics between brain cancer cells and neural stem cells, several research groups have succeeded to identify stem cell-like brain cancer cells (called "brain cancer stem cells") in brain tumors and brain cancer cell lines. The brain cancer stem cells, but not the other cancer cells, self-renew, form tumors when transplanted in vivo, and are highly resistant for both anti-cancer drugs and irradiation. Together all, these recent progresses suggest that it is crucial to characterize brain cancer stem cells and identify targets for the therapy. © 2008 Bentham Science Publishers Ltd.
• A potential role for bone morphogenetic protein signalling in glial cell fate determination following adult central nervous system injury in vivo

  David W. Hampton, Richard A. Asher, Toru Kondo, John D. Steeves, Matt S. Ramer, James W. Fawcett

  EUROPEAN JOURNAL OF NEUROSCIENCE 26 (11) 3024 - 3035 0953-816X 2007/12 [Refereed][Not invited]
   

  Bone morphogenetic proteins (BMPs) and their endogenous inhibitors, including noggin, chordin and follistatin, have roles in pattern formation and fate specification of neuronal and glial cells during nervous system development. We have examined their influence on glial reactions in the injured central nervous system (CNS). We show that penetrating injuries to the brain and spinal cord resulted in the upregulation of BMP-2/4, BMP-7, and noggin, with the latter being expressed almost exclusively by reactive astrocytes at the injury site, and we show that astrocytes in vitro produce noggin. As BMPs have been shown to drive cultured NG2-positive oligodendrocyte precursors (OPCs) towards a multipotential phenotype (type II astrocytes), we investigated the effects of inhibiting noggin with a function-blocking antibody (noggin-FbAb). In vitro, BMP-driven conversion of OPCs to type 2 astrocytes was inhibited by noggin, an effect that was reversed by noggin-FbAb. Noggin-FbAb also increased the number of type 2 astrocytes generated from cultured OPCs exposed to an astrocyte feeder layer, consistent with astrocytes producing both BMPs and noggin. In knife cut injuries in vivo, noggin-FbAb treatment resulted in an increase in the number of NG2-positive cells and small GFAP-positive cells in the injury site, and the appearance of glial cells with the morphological and antigenic characteristics of type 2 astrocytes (as generated in vitro), with coexpression of both GFAP and NG2. This potential conversion of inhibitory OPCs to type 2 astrocyte-like cells in vivo suggests that endogenous BMPs, unmasked by noggin antagonism, might be exploited to manipulate cell fate following CNS trauma.
• Inhibition of histone deacetylase activity induces developmental plasticity in oligodendrocyte precursor cells

  Costas A. Lyssiotis, John Walker, Chunlei Wu, Toru Kondo, Peter G. Schultz, Xu Wu

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 (38) 14982 - 14987 0027-8424 2007/09 [Refereed][Not invited]
   

  Recently, it was demonstrated that lineage-committed oligodendrocyte precursor cells (OPCs) can be converted to multipotent neural stem-like cells, capable of generating both neurons and glia after exposure to bone morphogenetic proteins. In an effort to understand and control the developmental plasticity of OPCs, we developed a high-throughput screen to identify novel chemical inducers of OPC reprogramming. Using this system, we discovered that inhibition of histone deacetylase (HDAC) activity in OPCs acts as a priming event in the induction of developmental plasticity, thereby expanding the differentiation potential to include the neuronal lineage. This conversion was found to be mediated, in part, through reactivation of sox2 and was highly reproducible at the clonal level. Further, genome-wide expression analysis demonstrated that HDAC inhibitor treatment activated sox2 and 12 other genes that identify or maintain the neural stem cell state while simultaneously silencing a large group of oligodendrocyte lineage-specific genes. This series of experiments demonstrates that global histone acetylation, induced by HDAC inhibition, can partially reverse the lineage restriction of OPCs, thereby inducing developmental plasticity.
• Stem cell-like cancer cells in cancer cell lines

  T. Kondo

  Cancer Biomarkers 3 (4-5) 245 - 250 1574-0153 2007 [Refereed][Invited]
   

  Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Moreover, a small number of cancer cells express stem cell markers, including CD133 and ATP-binding cassette transporters, by which the cells can pump out specific fluorescence dyes, such as Hoechst33342, as well as anti-cancer drugs, suggesting that either cancer cells resemble stem cells or cancers contain stem cell-like cancer cells, called "cancer stem cells (CSCs)". Using the common characteristics of tissue-specific stem cells, it was demonstrated that many types of tumors and cancer cell lines contain CSCs, which self-renew, express stem cell markers, and are tumorigenic. It was also shown that CSCs are resistant to anti-cancer drugs and irradiation. Thus CSCs might be a crucial target for the therapy. Because tumors contain CSCs and recruited normal stem cells, both of which contribute to tumorigenesis, it is difficult to separate CSCs from tumors. By contrast, cancer cell lines do not have any contaminating normal stem cells that quickly loose mulitpotentiality and differentiate in normal culture condition, suggesting that cancer cell lines could be an attractive alternative source of cells for CSC research. In this review I summarize the recent progress in CSC research using cancer cell lines. © 2007 IOS Press. All rights reserved.
• Epigenetic alchemy for cell fate conversion

  Toru Kondo

  CURRENT OPINION IN GENETICS & DEVELOPMENT 16 (5) 502 - 507 0959-437X 2006/10 [Refereed][Invited]
   

  Recent progress in neural stem cell research shows that a number of extrinsic factors and intracellular mechanisms, including epigenetic modifications, are involved in the self-renewal of neural stem cells and in neuronal and glial differentiation. Remarkably, there is increasing evidence that the remodeling of chromatin structure and the alteration of epigenetic marks, including histone methylation and acetylation and DNA methylation, can cause committed cells to convert from one fate to another, and such converted cells are functional when transplanted in vivo. Thus, epigenetic research might generate the alchemy required to convert any non-neural stem cells into functional neural stem cells, which are few and difficult to extract from the adult central nervous system.
• Multiple roles of Id4 in developmental myelination: Predicted outcomes and unexpected findings

  Mireya Marin-Husstege, Ye He, Jiadong Li, Toru Kondo, Fred Sablitzky, Patrizia Casaccia-Bonnefil

  GLIA 54 (4) 285 - 296 0894-1491 2006/09 [Refereed][Not invited]
   

  Myelination in the central nervous system is a complex process requiring the integration of oligodendrocyte progenitor differentiation and the coordinate expression of myelin genes. This study addresses the role of the helix-loop-helix protein Id4 in these two events. Overexpression of Id4 in oligodendrocyte progenitors prevents differentiation and consequently decreases the endogenous expression of all myelin genes. Conversely, progenitors lacking Id4 display precocious differentiation both in vitro and in vivo, and this phenotype is partially compensated by increased apoptosis. Besides this role, Id4 also has the ability to decrease the activity of specific myelin promoters, since Id4 overexpression decreases the activity of luciferase reporter genes driven by the ceramide galactosyltransferase (CGT) or myelin basic protein (MBP) promoter, but not by a myelin proteolipid protein (PLP) promoter. Consistent with these results, the expression levels of MBP and CGT are greater in neonatal Id4 null mice when compared with wild-type siblings and correlate with the early detection of MBP immunoreactive myelinated fibers. In contrast, the levels of other myelin proteins, such as PLP and myelin associated glycoprotein (MAG) are decreased in the Id4 null mice. MAG expression is localized to the soma rather than the fibers of immunoreactive cells in the neonatal brain and compensated at later developmental stages. These data support the role of Id4 as oligodendrocyte differentiation inhibitor with the ability to differentially regulate the expression and subcellular distribution of myelin gene products. (c) 2006 Wiley-Liss, Inc.
• Brain cancer stem-like cells

  T Kondo

  EUROPEAN JOURNAL OF CANCER 42 (9) 1237 - 1242 0959-8049 2006/06 [Refereed][Invited]
   

  Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Moreover, many tumours and cancer cell lines express stem cell markers, including adenosine triphosphate (ATP)-binding cassette transporters, by which the cells pump out specific fluorescent dyes as well as anti-cancer drugs, suggesting either that cancer cells resemble stem cells or that cancers contain stem-like cells. Using the common characteristics of brain tumour cells and neural stem cells, several research groups have succeeded in identifying stem-like cells (cancer stem-like cells) in brain tumours and brain cancer cell lines. The purified cancer stem-like cells, but not the other cancer cells, self-renew and form tumours when transplanted in vivo. Thus, cancer stem-like cells in brain tumours might be a crucial target for anti-brain tumour therapy. (c) 2006 Elsevier Ltd. All rights reserved.
• [Cancer stem cells in cancer cell lines].

  Setoguchi T, Kondo T, Taga T

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 15 50 (15) 1995 - 2000 0039-9450 2005/12 [Refereed][Not invited]
  
• Id4 is required for the correct timing of neural differentiation

  L Bedford, R Walker, T Kondo, van Cruchten, I, ER King, F Sablitzky

  DEVELOPMENTAL BIOLOGY 280 (2) 386 - 395 0012-1606 2005/04 [Refereed][Not invited]
   

  Complex intrinsic and extrinsic mechanisms determine neural cell fate during development of the nervous system. Using Id4 deficient mice, we show that Id4 is required for normal development of the central nervous system (CNS), timing neural differentiation in the developing forebrain. In the absence of Id4, the ventricular zone of the neocortex, future hippocampus as well as lateral and medial ganglionic eminences exhibited a 20-30% reduction in mitotic neural precursor cells (NPCs). Although the number of apoptotic cells was significantly increased, the neocortex of Id4(-/-) embryos was consistently thicker due to premature neuronal differentiation, which resulted in an increase in early-bom neurons in the adult Id4(-/-) cortex. Late-bom cortical neurons and astrocytes in the cortex, septum, hippocampus and caudate putamen of Id4(-/-) adult brains were decreased, however, likely due to the depletion of the NPC pool. Consequently, adult ld4(-/-) brains were smaller and exhibited enlarged ventricles. In vitro analysis of neurosphere cultures revealed that proliferation of Id4-deficient NPCs was impaired and that BMP2-mediated astrocyte differentiation was accelerated in the absence of Id4. Together, these in vivo and in vitro data suggest a crucial role for Id4 in regulating NPC proliferation and differentiation. (c) 2005 Elsevier Inc. All rights reserved.
• Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

  T Kondo, M Raff

  GENES & DEVELOPMENT 18 (23) 2963 - 2972 0890-9369 2004/12 [Refereed][Not invited]
   

  We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm.
• Nuclear export of OLIG2 in neural stem cells is essential for ciliary neurotrophic factor-induced astrocyte differentiation

  T Setoguchi, T Kondo

  JOURNAL OF CELL BIOLOGY 166 (7) 963 - 968 0021-9525 2004/09 [Refereed][Not invited]
   

  Neural stem cell (NSC) differentiation is precisely controlled by a network of transcription factors, which themselves are regulated by extracellular signals (Bertrand, N., D.S. Castro, and F. Guillemot. 2002. Nat. Rev. Neurosci. 3:517-530; Shirasaki, R., and S.L. Pfaff. 2002. Annu. Rev. Neurosci. 25:251-281). One way that the activity of such transcription factors is controlled is by the regulation of their movement between the cytosol and nucleus (Vandromme, M., C. Gauthier-Rouviere, N. Lamb, and A. Fernandez. 1996. Trends Biochem. Sci. 21:59-64; Lei, E.P., and P.A. Silver. 2002. Dev. Cell. 2:261-272). Here we show that the basic helix-loop-helix transcription factor OLIG2, which has been shown to be required for motor neuron and oligodendrocyte development, is found in the cytoplasm, but not the nucleus, of astrocytes in culture and of a subset of astrocytes in the subventricular zone. We demonstrate that the accumulation of OLIG2 in the nucleus of NSCs blocks the CNTF-induced astrocyte differentiation and that the translocation of OLIG2 to the cytoplasm is promoted by activated AKT. We propose that the AKT-stimulated export of OLIG2 from the nucleus of NSCs is essential for the astrocyte differentiation.
• Cancer stem cells persist in many cancer cell lines

  T Setoguchi, T Taga, T Kondo

  CELL CYCLE 3 (4) 414 - 415 1538-4101 2004/04 [Refereed][Invited]
   

  Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells ( cancer stem cells). To help resolve this paradox, we investigated whether established malignant cell lines, which have been maintained over years in culture, contain a subpopulation of stem cells. We have shown that four cancer cell lines contain a small side population (SP), which, in many normal tissues, is enriched for stem cells of the tissue. We have also shown that SP cells in C6 glioma cell line, but not non-SP cells, can generate both SP and non-SP cells in culture and are largely responsible for the in vivo malignancy of this cell line. We propose that many cancer cell lines contain a minor subpopulation of stem cells that is enriched in a SP, can be maintained indefinitely in culture, and is crucial for their malignancy.
• A role for Noggin in the development of oligodendrocyte precursor cells

  T Kondo, MC Raff

  DEVELOPMENTAL BIOLOGY 267 (1) 242 - 251 0012-1606 2004/03 [Refereed][Not invited]
   

  Oligodendrocyte precursor cells (OPCs) can be differentiated in culture into either oligodendrocytes or type-2 astrocytes (2As), depending on the culture conditions. Whereas oligodendrocyte development can occur in the absence of inducing signals, 2A development apparently cannot. Fetal calf serum (FCS) and bone morphogenetic proteins (BMPs) are powerful inducers of 2A development in culture, but there is no compelling evidence that OPCs develop into astrocytes in vivo. We show here that BMPs are made by glial cells in the developing rat optic nerve, raising the question of why 2As do not normally develop in the optic nerve. We demonstrate that the BMP antagonist Noggin is strongly expressed by both OPCs and type-1 astrocytes in the developing optic nerve. We also show that depletion of Noggin by a small interference RNA inhibits OPC proliferation and induces 2A differentiation in the presence of a low, non-2A-inducing concentration of FCS. By contrast, enforced expression of Noggin in OPCs blocks FCS-induced 2A differentiation. These findings suggest that BMPs in FCS are largely responsible for the 2A-inducing activity of FCS and that Noggin may normally inhibit the formation of 2As in the developing CNS. (C) 2003 Published by Elsevier Inc.
• Negative regulatory effect of an oligodendrocytic bHLH factor OLIG2 on the astrocytic differentiation pathway

  S Fukuda, T Kondo, H Takebayashi, T Taga

  CELL DEATH AND DIFFERENTIATION 11 (2) 196 - 202 1350-9047 2004/02 [Refereed][Not invited]
   

  In the developing vertebrate nervous system, multipotent neural stem cells produce both neurons and glia. OLIG2 is a basic helix-loop-helix transcription factor that plays critical roles in oligodendrocyte and motor neuron development; however, its role in astrocytic development remains elusive. In this study, we analyzed an effect of OLIG2 on cytokine-induced astrocytic differentiation from mouse telencephalic neuroepithelial cells. We show that the presence of OLIG2 protein leads to inhibition of the promoter activation of astrocyte-specific glial fibrillary acidic protein gene. We found that OLIG2 abolishes complex formation between a transcriptional coactivator p300 and a transcription factor, signal transducer and activator of transcription 3 (STAT3), which is activated by astrocytic differentiation-inducing cytokines, such as leukemia inhibitory factor (LIF). The enforced expression of OLIG2 in neuroepithelial cells inhibits the LIF-induced astrocytic differentiation. We also show that the OLIG2 protein in the nuclei of neural precursor cells disappears in accordance with astrocytic differentiation during culture with LIF. Together, these results reveal a novel molecular function of OLIG2 on the astrocyte development.
• Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line

  T Kondo, T Setoguchi, T Taga

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (3) 781 - 786 0027-8424 2004/01 [Refereed][Not invited]
  
• Reversion of oligodendrocyte precursor cells to multipotent neural stem cells.

  Toru Kondo

  Biotech Medicine 20 4 - 6 2001 [Refereed][Invited]
  
• Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells

  T Kondo, M Raff

  SCIENCE 289 (5485) 1754 - 1757 0036-8075 2000/09 [Refereed][Not invited]
   

  During animal development, cells become progressively more restricted in the cell types to which they can give rise. In the central nervous system (CNS), for example, multipotential stem cells produce various kinds of specified precursors that divide a limited number of times before they terminally differentiate into either neurons OF glial cells. We show here that certain extracellular signals can induce oligodendrocyte precursor cells to revert to multipotential neural stem cells, which can self-renew and give rise to neurons and astrocytes, as well as to oligodendrocytes, Thus, these precursor cells have greater developmental potential than previously thought.
• Basic helix-loop-helix proteins and the timing of oligodendrocyte differentiation

  T Kondo, M Raff

  DEVELOPMENT 127 (14) 2989 - 2998 0950-1991 2000/07 [Refereed][Not invited]
   

  An intracellular timer in oligodendrocyte precursor cells is thought to help control the timing of their differentiation. We show here that the expression of the Hes5 and Mash1 genes, which encode neural-specific bHLH proteins, decrease and increase, respectively, in these cells with a time course expected if the proteins are part of the timer. We show that enforced expression of Hes5 in purified precursor cells strongly inhibits the normal increase in the thyroid hormone receptor protein TR beta 1, which is thought to be part of the timing mechanism; it also strongly inhibits the differentiation induced by either mitogen withdrawal or thyroid hormone treatment. Enforced expression of Mash1, bg contrast, somewhat accelerates the increase in TR beta 1 protein. These findings suggest that Hes5 and Mash1 may be part of the cell-intrinsic timer in the precursor cells.
• Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

  A Eguchi, T Kondoh, H Kosaka, T Suzuki, H Momota, A Masago, T Yoshida, H Taira, A Ishii-Watabe, J Okabe, JH Hu, N Miura, S Ueda, Y Suzuki, T Taki, T Hayakawa, M Nakanishi

  JOURNAL OF BIOLOGICAL CHEMISTRY 275 (23) 17549 - 17555 0021-9258 2000/06 [Refereed][Not invited]
   

  In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.
• The Id4 HLH protein and the timing of oligodendrocyte differentiation

  T Kondo, M Raff

  EMBO JOURNAL 19 (9) 1998 - 2007 0261-4189 2000/05 [Refereed][Not invited]
   

  tin intracellular timer is thought to help control the timing of oligodendrocyte differentiation. We show here that the expression of the helix-loop-helix gene Id4 in oligodendrocyte precursor cells decreases irt vivo and in vitro with a time course expected if Id4 is part of the timer. We also show that Id4 expression decreases prematurely when the precursor cells are induced to differentiate by mitogen withdrawal, Both Id4 mRNA and protein decrease together under all of these conditions, suggesting that the control of Id4 expression is transcriptional. Finally, we show that enforced expression of Id4 stimulates cell proliferation and blocks differentiation induced by either mitogen withdrawal or treatment with thyroid hormone. These findings suggest that a progressive fall in Id4 transcription is part of the intracellular timer that helps determine when oligodendrocyte precursor cells withdraw from the cell cycle and differentiate.
• Activation of distinct caspase-like proteases by Fas and reaper in Drosophila cells

  T Kondo, T Yokokura, S Nagata

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 94 (22) 11951 - 11956 0027-8424 1997/10 [Refereed][Not invited]
   

  The cytoplasmic region of Fas, a mammalian death factor receptor, shares a limited homology with reaper, an apoptosis-inducing protein in Drosophila, Expression of either the Fas cytoplasmic region (FasC) or of reaper in Drosophila cells caused cell death, The death process induced by FasC or reaper was inhibited by crmA or p35, suggesting that its death process is mediated by caspase-like proteases, Both Ac-YVAD aldehyde and Ac-DEVD aldehyde, specific inhibitors of caspase 1- and caspase 3-like proteases, respectively, inhibited the FasC-induced death of Drosophila cells, However, the cell death induced by reaper was inhibited by Ac-DEVD aldehyde, but not by Ac-YVAD aldehyde. A caspase 1-like protease activity that preferentially recognizes the YVAD sequence gradually increased in the cytosolic fraction of the FasC-activated cells, whereas the caspase 3-like protease activity recognizing the DEVD sequence was observed in the reaper-activated cells, Partial purification and biochemical characterization of the proteases indicated that there are at least three distinct caspase-like proteases in Drosophila cells, which are differentially activated by FasC and reaper, The conservation of the Fas-death signaling pathway in Drosophila cells, which is distinct from that for reaper, may indicate that cell death in Drosophila is controlled not only by the reaper suicide gene, but also by a Fas-like killer gene.
• Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain

  H Mizuguchi, T Nakagawa, Y Morioka, S Imazu, M Nakanishi, T Kondo, T Hayakawa, T Mayumi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234 (1) 15 - 18 0006-291X 1997/05 [Refereed][Not invited]
   

  Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL. In addition, in vitro experiments using LLCMK2 cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection, In contrast, pRSVL required 24-48 h for induction of luciferase expression. Our results suggest that the cytoplasmic gene expression system is useful for gene delivery into the CNS. (C) 1997 Academic Press.
• Essential roles of the Fas ligand in the development of hepatitis

  T Kondo, T Suda, H Fukuyama, M Adachi, S Nagata

  NATURE MEDICINE 3 (4) 409 - 413 1078-8956 1997/04 [Refereed][Not invited]
   

  The Fas ligand (FasL) is expressed in activated T cells and induces apoptosis in Fas-bearing cells. A cytotoxic T lymphocyte (CTL) clone specific for hepatitis B surface antigen (HBsAg) causes an acute liver disease in HBsAg transgenic mice. Here we observed that the CTL clone killed hepatocytes expressing HBsAg in a Fas-dependent manner. Administration of the soluble form of Fas into HBsAg transgenic mice prevented the CTL-induced liver disease. In the second model, mice were primed with Propionibacterium acnes. A subsequent challenge with lipopolysaccharide (LPS) killed the mice by inducing liver injury. Neutralization of FasL rescued the mice from LPS-induced mortality, and Fas-null mice were resistant to LPS-induced mortality. These results suggest that FasL has an essential role in the development of hepatitis.
• Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense

  A Kato, Y Sakai, T Shioda, T Kondo, M Nakanishi, Y Nagai

  GENES TO CELLS 1 (6) 569 - 579 1356-9597 1996/06 [Refereed][Not invited]
   

  Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome, Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA, Starting with genomic negative sense (-) RNA has been unsuccessful, Furthermore, the recovery efficiency has often been extremely low. Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (-)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (-)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5' ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein. Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (-)RNA template, Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.
• TARGETED MUTATION IN THE FAS GENE CAUSES HYPERPLASIA IN PERIPHERAL LYMPHOID ORGANS AND LIVER

  M ADACHI, S SUEMATSU, T KONDO, J OGASAWARA, T TANAKA, N YOSHIDA, S NAGATA

  NATURE GENETICS 11 (3) 294 - 300 1061-4036 1995/11 [Refereed][Not invited]
   

  Fas, a type I membrane protein that transduces an apoptotic signal, is expressed in lymphocytes as well as in various tissues such as the liver, lung and heart. The mouse lymphoproliferation (lpr) mutation is a leaky mutation in Fas. By means of gene targeting, we generated a mouse strain which is completely deficient in Fas. In addition to the massive production of lymphocytes, the Fas-null mice showed substantial liver hyperplasia, which was accompanied by the enlargement of nuclei in hepatocytes. The Fas system seems to play a role in the apoptotic process to maintain homeostasis of the liver as well as the peripheral lymphoid organs.
• A NOVEL GENE-TRANSFER TECHNIQUE MEDIATED BY HVJ (SENDAI VIRUS), NUCLEAR-PROTEIN, AND LIPOSOMES

  N TOMITA, J HIGAKI, T OGIHARA, T KONDO, Y KANEDA

  CANCER DETECTION AND PREVENTION 18 (6) 485 - 491 0361-090X 1994 [Refereed][Not invited]
   

  There have been many reports of in vivo direct gene transfer methods, but there are problems with these methods. Recently, we developed a novel, nontoxic and efficient in vivo direct gene transfer method mediated by Sendai virus (HVJ) and liposomes. In our system, foreign genes and nuclear proteins were encapsulated into the same Liposomes, which were then treated with inactivated HVJ. The properties of our system are that HVJ enables foreign genes to be introduced directly into the cytoplasm by membrane fusion and that nuclear proteins transport the foreign genes rapidly into the nuclei. In the present study, we succeeded in introducing and expressing the functional gene for human insulin, and we observed the expression of this gene in mouse plasma and the reduction of plasma glucose. Our system will provide a new method of gene transfer aimed at postnatal gene therapy in various diseases.
• TEMPERATURE-SENSITIVE PHENOTYPE OF A MUTANT SENDAI VIRUS-STRAIN IS CAUSED BY ITS INSUFFICIENT ACCUMULATION OF THE M-PROTEIN

  T KONDO, T YOSHIDA, N MIURA, M NAKANISHI

  JOURNAL OF BIOLOGICAL CHEMISTRY 268 (29) 21924 - 21930 0021-9258 1993/10 [Refereed][Not invited]
   

  We investigated the process interrupting the production of a temperature-sensitive mutant strain of Sendai virus, Cl.151, at the nonpermissive temperature (38-degrees-C). The amount of virus M protein increased up to 6-fold when the cells persistently infected with Cl.151 strain at 38-degrees-C are transferred to 32-degrees-C, while the amount of nucleocapsid proteins did not alter. Cl.151 strain could restore virus production at 38-degrees-C not only by the supplementation of M protein of wild type (Z) strain but also by the supplementation of M protein of Cl.151 strain. Neither the amount of M mRNA nor the rate of synthesis of M protein was altered by temperature in cells infected with the Cl.151 strain. However, we found that M protein of Cl.151 virus, which has 3-amino acid alterations from the wild type, was highly unstable at 38-degrees-C when expressed under the control of an actin promoter. These results clearly show that Sendai virus M protein has a critical role in the production of virus particles without affecting virus gene expression.

Books etc

• 神経膠芽腫幹細胞膜タンパク質Ceacam1LとEva1

  近藤亨 (Single work)
  細胞 2016
• グリオブラストーマ幹細胞の基礎研究からの新規治療法探索

  近藤亨 (Single work)
  Bio Express 2016
• 神経膠芽腫幹細胞とマイクロRNA

  近藤亨 (Single work)
  医学のあゆみ、医歯薬出版 2014
• グリオーマ幹細胞特異的因子群を標的とした新規治療法の開発

  近藤亨 
  次世代がん戦略研究update がん基盤生物学ー革新的シーズ育成に向けてー 2013
• 癌幹細胞を標的とした治療のこれからーグリオーマ幹細胞をモデルとして

  近藤亨 (Single work)
  Frontiers in Gastroenterology、メディカルレビュー社 2013
• モデル動物利用マニュアル「疾患モデルの作製と利用－がん」

  近藤亨 (Single workグリオーマ)
  エル・アイ・シー社 2012
• オリゴデンドロサイト前駆細胞

  近藤亨 (Single work)
  細胞工学 2012
• Stem Cells and Cancer Stem Cells

  Kondo, T (Single workTumorigenesis of glioma-initiating cells: Role of Sox11.)
  Springer 2011
• Advances in Cancer Stem Cell Biology

  Kondo, T (Single workStem cells and cancer stem cells –new insights.)
  Springer 2011
• 脳腫瘍の癌幹細胞治療戦略

  近藤亨 (Single work)
  医薬ジャーナル 2011
• 脳腫瘍のCSC

  近藤亨 (Single work)
  BIO Clinica 2011
• グリオーマ

  近藤亨 (Single work)
  臨床検査 2011
• 膠芽腫

  近藤亨 (Single work)
  Clinical Neuroscience、中外医学社 2010
• 幹細胞生物学からみた脳腫瘍の発生機序

  近藤亨 (Single work)
  新時代の脳腫瘍学—診断・治療の最前線— 日本臨床社 2010
• 人工グリオーマからの考察

  近藤亨 (Single work)
  再生医療、メディカルレビュー社 2010
• カラー図説：癌幹細胞と分子標的薬

  近藤亨 (Single work)
  固形癌の最新治療—癌治療への新たな取り組みー 日本臨床社 2010
• 炎症・再生医学事典

  近藤亨 (Single work癌幹細胞)
  朝倉書店 2009
• オリゴデンドロサイトの分化制御機構とオリゴデンドログリオーマの発生

  近藤亨 (Single work)
  Brain and Nerve 2009
• 癌幹細胞マーカー

  近藤亨 (Single work)
  Surgery Frontier 2009
• 脳腫瘍における癌幹細胞

  近藤亨 (Single work)
  実験医学別冊 癌と微小環境 2009
• シリーズ脳科学4、脳の発生と発達

  近藤亨 (Single work神経細胞分化と可塑性)
  東京大学出版 2008
• 脳腫瘍「癌幹細胞」研究の現状

  秀拓一郎, 近藤亨 (Joint work)
  実験医学、羊土社 2008
• 癌幹細胞¬−癌研究のフロンティア

  近藤亨 (Single work)
  実験医学、羊土社 2008
• グリオーマ幹細胞

  近藤亨 (Single work)
  細胞 2008
• 固形がんにおけるがん幹細胞研究の進展

  近藤亨 
  血液・腫瘍化 2008
• 幹細胞様グリオーマ細胞

  近藤亨 (Single work)
  医学のあゆみ、医歯薬出版 2008
• グリオーマ起源細胞

  近藤亨 (Single work)
  Clinical neuroscience、中外医学社 2008
• Cancer stem cell marker

  Toru Kondo (Single work)
  Cancer Frontier 2008
• 腫瘍発生とエピジェネテック

  近藤亨 (Single work)
  分子細胞治療、先端医学社 2007
• グリオーマ細胞株に存在する幹細胞様細胞

  近藤亨 (Single work)
  医学のあゆみ、医歯薬出版 2006
• 幹細胞様ガン細胞

  近藤亨 (Single work)
  Cancer Frontier 2006
• 脳腫瘍に内在する幹細胞様細胞

  近藤亨 (Single work)
  再生医療、メディカルレビュー社 2006
• 癌幹細胞の単離と制御シグナル〜真の癌幹細胞分離へのアプローチ

  近藤亨 (Single work)
  実験医学、 羊土社 2006
• オリゴデンドロサイト前駆細胞の脱分化とエピジェネティクス変化

  近藤亨 (Single work)
  実験医学別冊、 羊土社 2006
• オリゴデンドロサイト前駆細胞の可塑性

  近藤亨 (Single work)
  実験医学、羊土社 2004
• オリゴデンドロサイトの分化制御とその可塑性

  近藤亨 (Single work)
  分子細胞治療、先端医学社 2002
• 組織幹細胞の可塑性

  近藤亨 
  再生医学・再生医療、現代化学増刊、東京化学同人 2002
• オリゴデンドロサイトの発生を制御する細胞内在時計

  近藤亨 
  医学のあゆみ、医歯薬出版 2002
• グリア前駆細胞の可塑性

  近藤亨 
  Clinical neuroscience、中外医学社 2002
• 神経幹細胞の分化制御機構

  近藤亨 
  炎症と免疫、先端医学社 2002
• ラットオリゴデンドロサイト前駆細胞の単離法

  近藤亨 
  実験医学、羊土社 2001
• Timing cell-cycle exit and differentiation in oligodendrocyte development

  Raff, M, Apperly, J, Kondo, T, Tokumoto, Y, Tang, D (Joint work)
  Novartis Found Symp. 2001
• オリゴデンドロサイトの発生を制御する“細胞時計”

  近藤亨 (Single work)
  蛋白質核酸酵素、共立出版 2001
• 神経発生の逆行；オリゴデンドロサイト前駆細胞から神経幹細胞へ

  近藤亨 (Single work)
  実験医学、羊土社 2000
• 肝炎発症におけるFas/Fasリガンドの働き

  近藤亨 
  免疫 Immunology Frontier、メディカルビュー社 1998
• 生物薬科学実験講座 第３巻

  中西真人, 芦原賢一, 真弓忠範, 近藤 亨, 米田悦啓 (Joint workHVJエンベロープタンパク質を用いた細胞内導入法)
  広川書店 1995
• Fasリガンド

  近藤亨 
  免疫 Immunology Frontier、メディカルビュー社 1995
• Gene Introduction into animal tissues

  (Joint work)
  Harwood Academic Publishers Gmbh 1995
• 膜融合リポソームを使った細胞への高能率遺伝子導入

  中西真人, 芦原賢一, 千田隆夫, 近藤 亨, 真弓忠範 (Joint work)
  実験医学、羊土社 1994
• 標的臓器への遺伝子導入による治療

  中西真人, 近藤 亨, 芦原賢一 (Joint work)
  nanoGIGA、日本臨床社 1992
• 動物個体臓器への遺伝子導入

  中西真人, 加藤啓子, 近藤 亨 (Joint work)
  代謝別冊 中山書店 1990

MISC

• 腫瘍開始細胞は炎症環境を形成し免疫細胞を老化させて抗腫瘍応答の低下を導くことで免疫健常個体における腫瘍発生を許容させる

  和田 はるか, 近藤 亨, 清野 研一郎  日本癌学会総会記事  79回-  OJ12  -8  2020/10
• がん幹細胞はそれ自身が原炎症性細胞であり微小環境に免疫老化及び免疫抑制をもたらすことで健常動物における造腫瘍能を担保する(Tumor-initiating cell induce immuno-hyporesponsiveness following cellular senescence to Mφs guarantee its tumorigenesis)

  和田 はるか, バグダーディー・ムハンマド, 近藤 亨, 清野 研一郎  日本癌学会総会記事  78回-  J  -1042  2019/09
• がん幹細胞はそれ自身が原炎症性細胞であり微小環境に免疫老化及び免疫抑制をもたらすことで健常動物における造腫瘍能を担保する

  和田 はるか, 村田 智己, 大塚 亮, 森口 徹夫, バグダーディー・ムハンマド, 近藤 亨, 清野 研一郎  日本がん免疫学会総会プログラム・抄録集  23回-  131  -131  2019/07
• 血漿エクソソーム由来microRNAを用いたグリオブラストーマ診断バイオマーカーの探索

  山口 響子, 森口 徹生, 山下 大介, 大西 丘倫, 金子 貞男, 的場 亮, 鄭 漢忠, 近藤 亨  北海道歯学雑誌  39-  (2)  94  -103  2019/03  

  最も悪性度の高い脳腫瘍の1つであるグリオブラストーマ(GBM)は、標準治療(外科手術および化学放射線療法)を施しても平均生存期間中間値(約15ヵ月)が極めて短い難治性疾患である。この難治性の原因の1つは、腫瘍細胞の強い組織浸潤能と増殖能により神経症状発症時には摘出不可能な範囲に腫瘍細胞が拡散しているためである。つまり、簡便かつ検出感度の高いGBM診断用バイオマーカーがあれば、早期腫瘍摘出が可能となり、予後の改善が期待できる。分泌小胞体エクソソームは、その産生細胞が発現しているmRNA、microRNA(miR)、タンパクなどを含み、様々な体液中に安定に存在することから、新たなバイオマーカー探索の標的としてその解析と利用が注目されている。今回、私たちはGBM患者と健常人の血漿中のエクソソームに含まれるmiRを比較分析することで、診断マーカーとなりうるmiRの同定を目的として解析を行った。GBM患者6人の凍結血漿8検体(同一患者の再発術前1検体と術後1検体を含む)と健常人2人の凍結血漿を用いて解析を行った。凍結血漿から調整したエクソソーム内miRについて、RNA-seqを用いた網羅的な解析を行い、GBM患者血漿エクソソームに多く含まれる34種のmiRと健常人血漿エクソソームに多く含まれる47種のmiRを同定した。GBM患者血漿エクソソームに多く含まれていたmiRの中で、エクソソームバイオマーカーとして報告のないmiR-186とmiR-20aについて、定量PCRを用いてGBM患者と健常人血漿エクソソーム内miR量を検討した。その結果、miR-20aはGBM患者と健常人間で有意な差は認められなかったが、GBM患者5人中4人の血漿エクソソームにmiR-186が豊富に含まれていることを確認した。加えて、術後患者ではmiR-186量が健常人レベルまで減少していることを発見した。さらに、標準治療を行った再発例においても、術前血漿エクソソーム内miR-186の上昇を認めた。これらの結果は、血漿エクソソーム内miR-186がGBMの病状に即した新規バイオマーカーである可能性を示唆している。(著者抄録)
• 免疫健常個体において造腫瘍能を発揮する腫瘍開始細胞の免疫学的因子の同定(Tumor initiating cell in immunocompetent animal defined by immunological features)

  和田 はるか, Baghdadi Muhammad, 森口 徹生, 近藤 亨, 清野 研一郎  日本癌学会総会記事  77回-  2327  -2327  2018/09
• Secreted tumor suppressor Ecrg4 acts as a novel immunosurveillance factor

  Tetsuo Moriguchi, Shuji Takeda, Uichi Koshimizu, Hidemitsu Kitamura, Toru Kondo  INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE  40-  S37  -S37  2017  [Not refereed][Not invited]
  
• グリオーマ幹細胞に特異的な発現を示すexosomal microRNAの同定

  山下 大介, 末廣 諭, 近藤 亨, 大西 丘倫  日本癌学会総会記事  75回-  P  -3093  2016/10  [Not refereed][Not invited]
  
• Glimはマウス及びヒトのグリオーマ形成能を亢進させる

  大津 直樹, 中谷 有香, 山下 大介, 大西 丘倫, 近藤 亨  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [4T18  -12(3P1074)]  2015/12  [Not refereed][Not invited]
  
• グリオーマ幹細胞の腫瘍形成を抑制するmicroRNA-340および標的分子PLATの同定と機能解析

  山下 大介, 近藤 亨, 大西 丘倫  日本癌学会総会記事  74回-  E  -1086  2015/10  [Not refereed][Not invited]
  
• Glim2 regulates the proliferation of glioblastoma-initiating cells through the activation of the STAT3 signaling

  Toru Kondo, Sadahiro Kaneko  INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE  36-  S33  -S33  2015  [Not refereed][Not invited]
  
• ヒトグリオーマ幹細胞の腫瘍形成を抑制するmicroRNA-340の同定 機能解析と標的分子の検索

  山下 大介, 近藤 亨, 大上 史朗, 高橋 寿明, 末廣 諭, 井上 明宏, 高野 昌平, 大西 丘倫  Brain Tumor Pathology  31-  (Suppl.)  110  -110  2014/05  [Not refereed][Not invited]
  
• ヒトグリオーマ幹細胞の腫瘍形成を抑制するmicroRNA‐340とその標的分子PLATの機能解析

  山下大介, 近藤亨, 末廣諭, 井上明宏, 高野昌平, 大上史朗, 大西丘倫  日本脳腫瘍学会プログラム・抄録集  32nd-  84  2014  [Not refereed][Not invited]
  
• IDENTIFICATION AND CHARACTERIZATION OF A NOVEL microRNA INVOLVED IN GLIOMAGENESIS

  Daisuke Yamashita, Toru Kondo, Hisaaki Takahashi, Akihiro Inoue, Shohei Kohno, Hironobu Harada, Shiro Ohue, Takanori Ohnishi  NEURO-ONCOLOGY  15-  31  -31  2013/11  [Not refereed][Not invited]
  
• グリオーマ形成に関わる新規microRNAの性状解析

  山下 大介, 近藤 亨, 高橋 寿明, 井上 明宏, 高野 昌平, 原田 広信, 大上 史朗, 久門 良明, 田中 潤也, 大西 丘倫  Brain Tumor Pathology  30-  (Suppl.)  124  -124  2013/05  [Not refereed][Not invited]
  
• 新規グリオーマ幹細胞膜タンパク質Glimの機能解析

  大津直樹, 中谷友香, 山下大介, 大西丘倫, 近藤亨  日本分子生物学会年会プログラム・要旨集(Web)  36th-  1P-0815 (WEB ONLY)  2013  [Not refereed][Not invited]
  
• Glioma形成に関わる新規microRNA(miR‐X)の機能解析及びその標的遺伝子の同定

  山下大介, 近藤亨, 大上史朗, 高橋寿明, 末廣諭, 井上明宏, 高野昌平, 大西丘倫  日本脳腫瘍学会プログラム・抄録集  31st-  190  2013  [Not refereed][Not invited]
  
• SOX11 PREVENTS TUMORIGENESIS OF GLIOMA-INITIATING CELLS

  Takuichiro Hide, Tatsuya Takezaki, Hideo Nakamura, Keishi Makino, Jun-ichi Kuratsu, Toru Kondo  NEURO-ONCOLOGY  12-  122  -122  2010/11  [Not refereed][Not invited]
  
• Induced glioma-initiating cell models reveal novel therapeutic targets

  Toru Kondo, Takuichiro Hide, Tatsuya Takezaki, Yuka Nakatani, Hideo Nakamura, Junichi Kuratsu  INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE  26-  S36  -S36  2010  [Not refereed][Not invited]
  
• ESTABLISHMENT OF ARTIFICIAL GLIOBLASTOMA INITIATING CELLS IN MOUSE

  Takuichiro Hide, Tatsuya Takezaki, Hideo Nakamura, Keishi Makino, Jun-ichi Kuratsu, Toru Kondo  NEURO-ONCOLOGY  11-  (6)  932  -932  2009/12  [Not refereed][Not invited]
  
• HEDGEHOG PATHWAY AND GLIOMAGENESIS

  Tatsuya Takezaki, Hideichiro Taku, Hideo Nakamura, Jun-ichi Kuratsu, Toru Kondo  NEURO-ONCOLOGY  11-  (6)  956  -956  2009/12  [Not refereed][Not invited]
  
• Functional analysis of neural stem cell factors in gliomas

  Toru Kondo, Tatsuya Takezaki, Hiromi Takanaga, Takuichiro Hide  NEUROSCIENCE RESEARCH  61-  S69  -S69  2008  [Not refereed][Not invited]
  
• 癌幹細胞の同定とその意義

  瀬戸口 啓夫, 近藤 亨, 田賀 哲也  蛋白質核酸酵素  50-  (15)  1995  -2000  2005/12
• A role for Noggin in the development of oligodendrocyte precursor cells (vol 267, pg 242, 2004)

  T Kondo, MC Raff  DEVELOPMENTAL BIOLOGY  270-  (1)  272  -272  2004/06  [Not refereed][Not invited]
  
• Sendai virus vectors: New systems for gene transfer and expression

  M Nakanishi, H Mizuguchi, K Ashihara, T Senda, E Nagoshi, T Akuta, T Kondoh, T Mayumi  JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION  21-  (1)  92  -94  1996/07  [Not refereed][Not invited]
  

Industrial Property Rights

• 特願2018-239318:多能性幹細胞を除去するための組成物、及び多能性幹細胞の除去方法  2018年/12

  近藤亨  北海道大学
• 特願2018-108728:グリオーマの処置剤および医薬組成物  2018年/06

  近藤亨, 石井由紀子  北海道大学, 富士フイルム
• 特願2017-21550:抗Eva1タンパク質抗体  2017年

  横山茂之, 村松知成, 寺田貴帆, 近藤亨
• 特願2015-213455:グリオーマ治療剤及びその有効性を判定する方法  2015年/10/29

  近藤亨
• 特願2015-152941:抗Eva1タンパク質抗体  2015年/07/31

  近藤亨
• 特願2015-15982:がん幹細胞に対する増殖抑制能を有するマイクロＲＮＡをスクリーニングする方法及びマイクロＲＮＡを有効成分とするがん幹細胞の増殖抑制剤  2015年/01/29

  近藤亨
• 特願2013-238279:グリオーマ形成阻害作用を有するmicroRNA  2013年/11/18

  近藤亨、的場亮、山下大介  近藤亨, DNAチップ研究所, 愛媛大学

Research Grants & Projects

• Positional and quantitative regulation of the central nervous cells by interaction of pattern formation, proliferation and differentiation timing

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2020/04 -2023/03 

  Author : 笹井 紀明, 磯谷 綾子, 近藤 亨, 別所 康全, 白井 学

   

  生物の各器官には、様々な機能を持つ細胞が位置的だけでなく量的（細胞数）にも正確に配置されており、それらが相互作用し合うことによって全体として特有の機能を発揮している。本研究の目的は、発生途上にある中枢神経系をモデルにして、シグナルの経時的な変化、時空間特異的な増殖と細胞の分化を司る分子を同定して機能解析し、組織内での多様な細胞分化・パターン形成の分子メカニズムを明らかにすることである。 2020年度は、神経前駆細胞を分化方向にスイッチする遺伝子の１つをスクリーニングにより同定し、主にニワトリ胚を用いてその機能を明らかにした。ニワトリ胚から単離した神経前駆細胞に経時的に遺伝子発現解析を行い、分化と同時に発現が惹起される遺伝子を網羅的に探索した結果、いくつかの分化制御因子が単離された。このうちの１つの転写因子Sox14は神経管が形成された直後から前駆細胞に発現し、神経前駆細胞の分化を一定段階まで促進することが明らかになった。さらに、同時期に同細胞で発現する別の転写因子Chx10との機能的差異を解析したところ、Sox14は神経分化を促進する役割を持つ一方、Chx10は細胞運命（分化方向）を決める因子であることが示唆された。このように２つの転写因子が相互的に働くことにより、分化が進行するメカニズムの一端が明らかになった。 同時に、Sox14のノックアウト細胞をマウスES細胞において作成し、マウスの神経分化でSox14が必須の役割を果たすことを証明した。
• Development of Innovative Gene-Virus Therapy for Tumor Lysis, Immunity Induction, and Cancer Stem Cell Suppression in Bone and Soft Tissue Sarcoma.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2017/04 -2020/03 

  Author : Kosai Ken-ichiro

   

  We generated novel Surv.m-CRA expressing various therapeutic genes and analyzed their in vitro expression and various functions (tumor lysis, systemic anti-tumor immunity induction, etc.). These experiments first confirmed the expression and function of the therapeutic genes in each of the novel Surv. m-CRA and then largely confirmed their therapeutic properties. In addition, we conducted treatment experiments on several novel Surv. m-CRA in in vivo animal models to confirm their therapeutic efficacy. Thus, therapeutic properties of each new Surv. m-CRA-1 as well as experimental data on its usefulness, have been obtained from time to time.
• miRNA制御Crispr/Cas9発現依存的にがん幹細胞機能因子群をゲノム編集する新規がん治療用ベクターの開発

  日本医療研究開発機構：革新的がん医療実現化研究事業

  Date (from‐to) : 2016/11 -2017/03 

  Author : 近藤亨
• グリオーマ幹細胞特異的因子群を標的とした新規治療法の開発

  文部科学省：次世代がん研究戦略推進プロジェクト

  Date (from‐to) : 2011 -2016 

  Author : 近藤亨
• 癌幹細胞を標的とした癌根絶療法の創出

  文部科学省：分子イメージング研究戦略推進プログラム

  Date (from‐to) : 2010/04 -2015/03 

  Author : 近藤亨
• 人工グリオーマ幹細胞エキソソームを利用した診断・治療標的検索マイクロアレイの開発

  科学技術振興機構：研究成果最適展開支援プログラム

  Date (from‐to) : 2011 -2012 

  Author : 近藤亨
• 癌幹細胞―正常細胞間コミュニーケーション分子の同定と性状解析

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2011 -2012 

  Author : 近藤 亨

   

  １、Ecrg4下流因子の同定とその機能解析 H23年度にEcrg4+/+グリオーマ幹細胞（GIC）の野生型マウス脳における腫瘍形成能が、Ecrg4-/-GICに比べ著しく弱い事が発見した。両GICはヌードマウス脳に腫瘍を形成する事から、Ecrg4は免疫細胞を活性化する新規癌抗原であると考えられた。H24年度は、Ecrg4がT・B細胞のどちらに働いているのかの検討を進めた。始めに、抗Ecrg4抗体がEcrg4+/+GICを移植した野生型マウス末梢血中に 存在するかについて検討した結果、検出感度以下であった。この結果は、液性免疫によるEcrg4+/+GICの腫瘍形成抑制の可能性は低いことを示唆している。T細胞／NK細胞による細胞傷害実験系の確立と癌抗原としてのEcrg4の働きについて検討する予定である。同時に、Ecrg4+/+、+/-と-/-神経幹細胞（NSC）とこれら細胞から誘導したGICの遺伝子発現プロファイルを調べ、NSCとGICのEcrg4下流因子群を抽出した。これら因子の働きについて今後検討を進める予定である。 ２、Ecrg4受容体の同定 Ecrg4-Fc融合タンパク質とcDNA発現ライブラリーを用いたスクリーニングにより抽出したEcrg4受容体（Ecrg4R）候補因子群からEcrg4Rを同定した。Ecrg4Rは様々な生物学的機能（疾患、細胞老化を含む）を有する細胞膜タンパク質である。様々なEcrg4とその受容体の欠損タンパク質を作製し、Ecrg4のカルボキシル末端とEcrg4Rの細胞外領域が結合領域であることを同定した。今後はEcrg4Rシグナル伝達について検討を進める予定である。 ３、グリオーマ幹細胞由来細胞老化誘導因子の同定 マウスGICとヒトGIC群の遺伝子発現プロファイル解析から、発現上昇している分泌タンパク質群を抽出した。それらの機能は今後検討を進める予定である。
• The development of conditionally replicating adenovirus targeting cancer stem cell for innovative treatment for pediatric cancer

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : KOSAI Kenichiro, MITSUI Kaoru, IRIE Rie, KONDO Toru

   

  Although pediatric carcinomas have differentclinical and biological features from those of adult carcinomas, gene therapy for pediatric carcinoma has not been well developed. The present study aims to develop m-CRAs (conditionally replicating adenovirus targeting with multiple tumor-specific factors) that specifically treat cancer stem cells, leading to innovative therapy against pediatric carcinoma. First, we have established the methods to enrich and purify cancer stem cells in soft tissue sarcoma, giloblastoma, hepatoblastoma et al. Second, we revealed that our original m-CRAs efficiently treat cancer stem cells in such pediatric carcinoma.
• Functional analysis of Sox11 and Plagl1 in glioma stem cells.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2010 -2012 

  Author : KONDO Toru, OHNISHI Takanori, KOMATSU Kenshi

   

  We have examined about the expression of Sox11 and Plagl1 and their functions in glioma stem cells (GSC) and neural stem cells (NSC) and found the following results. (1) Expression of Sox11 in GSC is not regulated by either NeuroD or Neurogenin, both of which induce Sox11 expression in neural differentiation. We have identified the DNA domain (200bp) that regulates Sox11 expression in GSC. (2) Plagl1 is involved in both the maintenance of stemness in NSC and their DNA repair. (3) We have established efficient methods for human GSC preparation and their maintenance and examined their expression profiles.
• ヒトグリオーマ細胞株に存在する幹細胞様細胞の精製および新規マーカー遺伝子の単離

  文部科学省：科学研究費補助金（特定領域研究）

  Date (from‐to) : 2006/04 -2008/03 

  Author : 近藤亨
• 中枢神経系発生における分化抑制転写因子Id4の機能解析

  文部科学省：科学研究費補助金（特定領域研究）

  Date (from‐to) : 2002/04 -2003/03 

  Author : 近藤亨
• アポト-シスの分子機構とその生理作用

  文部科学省：科学研究費補助金(重点領域研究, 特定領域研究(A))

  Date (from‐to) : 1994 -1999 

  Author : 長田 重一, 近藤 亨, 渡辺 大介, 須田 貴司, 江成 政人, 田中 正人, 福永 理己郎, 福山 英啓

   

  アポトーシスは動物の発生過程で産生される不要な細胞,害となる細胞を取り除く細胞死の過程であり、細胞・核の凝縮、断片化、染色体DNAのヌクレオソームの単位への切断をともなっている。私達は、昨年、カスパーゼ(システンプロテアーゼ)によって活性化されるDNase(CAD,caspase-activated DNase)とその阻害たんぱく質(ICAD,inhibitor of CAD)を精製、その遺伝子を単離し、アポトーシスにおけるDNA断片化の分子機構を明らかとした。すなわちCADは増殖している細胞中ではICADと複合体を形成しており、アポトーシスの刺激によりカスパーゼが活性化されると、カスパーゼはICADを切断、不活化し自由になったCADが染色体DNAを切断する。本年度、CAD,ICADの組換えたんぱく質を大腸菌や蚕細胞で合成し,CADは高い比活性を持つDNaseであること,ICADはCADの阻害たんぱく質としてばかりでなく、CADが合成される際,シャペロンとして作用することを示した。すなわち、無細胞系や動物細胞でCADはそれ単独では活性を持つたんぱく質としては合成されず,機能的なたんぱく質が合成されるにはICADの存在が必須であった。また、ICADは塩酸グアニヂンによってdenatureしたCADのrefolding過程を促進した。一方,カスパーゼによって切断される部位に変異を...

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• 2014年4月1日 

  - 

  2016年3月31日 

  遺伝子病制御研究所附属感染癌研究センター長
• 2016年4月1日 

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  2018年3月31日 

  遺伝子病制御研究所附属感染癌研究センター長
• 2018年4月1日 

  - 

  2019年3月31日 

  遺伝子病制御研究所附属感染癌研究センター長

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• 2024/03 - Today   Cancer Heterogeneity and Plasticity   Editorial board member
• 2007 - Today   Associate Editor