Updated on 2022/08/17

写真a

 
SEMBA, Kentaro
 
Affiliation
Faculty of Science and Engineering, School of Advanced Science and Engineering
Job title
Professor

Concurrent Post

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

  • Affiliated organization   Global Education Center

Research Institute

  • 2020
    -
    2022

    理工学術院総合研究所   兼任研究員

Degree

  • 理学博士

Professional Memberships

  •  
     
     

    日本分子生物学会

  •  
     
     

    日本がん転移学会

  •  
     
     

    日本がん分子標的治療学会

  •  
     
     

    日本癌学会

 

Research Areas

  • Tumor biology

  • Molecular biology

Research Interests

  • 分子腫瘍学

  • がん遺伝子

  • シグナル伝達

  • 転移

  • トランスポゾン

Papers

  • Establishment of reporter cells that respond to glucocorticoids by a transposon-mediated promoter-trapping system.

    Kosuke Ishikawa, Sakura Tamamura, Kentaro Semba, Shinya Watanabe

    European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences   162   105819 - 105819  2021.07  [Refereed]  [International journal]

     View Summary

    Previously, we had established a highly sensitive trap vector system for the efficient isolation of reporter cells for a certain condition of interest. In this study, we used this system to screen reporter cells that express the luciferase and enhanced green fluorescent protein genes in response to dexamethasone, a glucocorticoid receptor agonist to facilitate glucocorticoid signaling research. In total, 10 clones were isolated. The insertion sites of the trap vector were analyzed using 5' rapid amplification of cDNA ends (5' RACE), whereupon LPIN1, PKP2, and FKBP5 were identified as genes that were upregulated by the dexamethasone treatment. Specifically, PKP2 has not previously been focused as a gene that responds to glucocorticoids. The PKP2 mRNA was analyzed and induction of the endogenous gene was confirmed by real-time polymerase chain reaction. Given that PKP2 does not appear to have a consensus glucocorticoid response element (GRE) sequence, this reporter clone could supplement the current GRE-based reporter systems that are prevalently used. Because different clones showed different responses to glucocorticoids, these clones should provide more information than analysis with a single reporter clone. This paper demonstrates that the previously developed trap vector technology can contribute to the rapid construction of drug evaluation systems.

    DOI PubMed

  • A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap.

    Honami Miyakura, Mei Fukuda, Hiroya Enomoto, Kosuke Ishikawa, Shinya Watanabe, Kentaro Semba

    PloS one   16 ( 5 ) e0251240  2021  [Refereed]  [International journal]

     View Summary

    We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system. This system requires creation of a bait strain that stably expresses a fusion product of part of the fluorescent monomeric Kusabira-Green (mKG) protein to a protein of interest. A PiggyBac transposon vector is then introduced into this strain, and a sequence encoding the remainder of mKG is inserted into the genome and fused randomly with endogenous genes. The binding partner can be identified by isolating cells that fluoresce when BiFC occurs. Using this system, we screened for interactors of p65 (also known as RELA), an NF-κB subunit, and isolated a number of mKG-positive clones. 5'- or 3'-RACE to produce cDNAs encoding mKG-fragment fusion genes and subsequent reconstitution assay identified PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP as p65 interactors. All of these, with the exception of CACYBP, are known regulators of NF-κB. Immunoprecipitation assay confirmed endogenously expressed CACYBP and p65 formed a complex. A reporter assay revealed that CACYBP enhanced 3κB reporter activation under TNFα stimulation. This screening system therefore represents a valuable method for identifying interacting factors that have not been identified by other methods.

    DOI PubMed

  • Proliferative Classification of Intracranially Injected HER2-positive Breast Cancer Cell Lines

    Yuka Kuroiwa, Jun Nakayama, Chihiro Adachi, Takafumi Inoue, Shinya Watanabe, Kentaro Semba

    Cancers   12 ( 7 ) 1811 - 1811  2020.07  [Refereed]

     View Summary

    HER2 is overexpressed in 25–30% of breast cancers, and approximately 30% of HER2-positive breast cancers metastasize to the brain. Although the incidence of brain metastasis in HER2-positive breast cancer is high, previous studies have been mainly based on cell lines of the triple-negative subtype, and the molecular mechanisms of brain metastasis in HER2-positive breast cancer are unclear. In the present study, we performed intracranial injection using nine HER2-positive breast cancer cell lines to evaluate their proliferative activity in brain tissue. Our results show that UACC-893 and MDA-MB-453 cells rapidly proliferated in the brain parenchyma, while the other seven cell lines moderately or slowly proliferated. Among these nine cell lines, the proliferative activity in brain tissue was not correlated with either the HER2 level or the HER2 phosphorylation status. To extract signature genes associated with brain colonization, we conducted microarray analysis and found that these two cell lines shared 138 gene expression patterns. Moreover, some of these genes were correlated with poor prognosis in HER2-positive breast cancer patients. Our findings might be helpful for further studying brain metastasis in HER2-positive breast cancer.

    DOI

  • Establishment and characterization of highly osteolytic luminal breast cancer cell lines by intra-caudal arterial injection

    Han Y, Nakayama J, Hayashi Y, Jeong S, Futakuchi M, Ito E, Watanabe S, Semba K

    Genes to Cells   25 ( 2 ) 111 - 123  2020.02  [Refereed]

    DOI PubMed

  • Genetic manipulation of the mammary gland and potential applications.

    Hiroaki Tagaya, Kentaro Semba, Kosuke Ishikawa

    Oncotarget   10 ( 42 ) 4253 - 4254  2019.07  [International journal]

    DOI PubMed

  • A method of producing genetically manipulated mouse mammary gland.

    Hiroaki Tagaya, Kosuke Ishikawa, Yoshito Hosokawa, Shun Kobayashi, Yukino Ueoka, Mayuna Shimada, Yasuko Ohashi, Hirofumi Mikami, Mizuki Yamamoto, Tatsuya Ihara, Kentaro Kumazawa, Kosuke Sugihara, Naoki Goshima, Shinya Watanabe, Kentaro Semba

    Breast cancer research : BCR   21 ( 1 ) 1 - 1  2019.01  [Refereed]  [International journal]

     View Summary

    BACKGROUND: To obtain a deep understanding of the mechanism by which breast cancer develops, the genes involved in tumorigenesis should be analyzed in vivo. Mouse mammary gland can regenerate completely from a mammary stem cell (MaSC), which enables us to analyze the effect of gene expression and repression on tumorigenesis in mammary gland regenerated from genetically manipulated MaSCs. Although lentiviral and retroviral systems have usually been applied for gene transduction into MaSCs, they are associated with difficulty in introducing long, repeated, or transcriptional termination sequences. There is thus a need for an easier and quicker gene delivery system. METHODS: We devised a new system for gene delivery into MaSCs using the piggyBac transposon vectors and electroporation. Compared with viral systems, this system enables easier and quicker transfection of even long, repeated, or transcriptional termination DNA sequences. We designed gene expression vectors of the transposon system, equipped with a luciferase (Luc) expression cassette for monitoring gene transduction into regenerative mammary gland in mice by in-vivo imaging. A doxycycline (Dox)-inducible system was also integrated for expressing the target gene after mammary regeneration to mimic the actual mechanism of tumorigenesis. RESULTS: With this new gene delivery system, genetically manipulated mammary glands were successfully reconstituted even though the vector size was > 200 kb and even in the presence of DNA elements such as promoters and transcription termination sequences, which are major obstacles to viral vector packaging. They differentiated correctly into both basal and luminal cells, and showed normal morphological change and milk production after pregnancy, as well as self-renewal capacity. Using the Tet-On system, gene expression can be controlled by the addition of Dox after mammary reconstitution. In a case study using polyoma-virus middle T antigen (PyMT), oncogene-induced tumorigenesis was achieved. The histological appearance of the tumor was highly similar to that of the mouse mammary tumor virus-PyMT transgenic mouse model. CONCLUSIONS: With this system, gene transduction in the mammary gland can be easily and quickly achieved, and gene expression can be controlled by Dox administration. This system for genetic manipulation could be useful for analyzing genes involved in breast cancer.

    DOI PubMed

  • A highly sensitive trap vector system for isolating reporter cells and identification of responsive genes.

    Kosuke Ishikawa, Yuta Kobayashi, Yutaro Wakabayashi, Shinya Watanabe, Kentaro Semba

    Biology methods & protocols   3 ( 1 ) bpy003  2018  [Refereed]  [International journal]

     View Summary

    We devised a versatile vector system for efficient isolation of reporter cells responding to a certain condition of interest. This system combines nontoxic GAL4-UAS and piggyBac transposon systems, allowing application to mammalian cells and improved expression of a fluorescent reporter protein for cell sorting. Case studies under conditions of c-MYC gene induction or endoplasmic reticulum (ER) stress with thapsigargin on mouse or human cell lines confirmed easy and efficient isolation of responsive reporter cells. Sequence analyses of the integrated loci of the thapsigargin-responsive clones identified responsive genes including BiP and OSBPL9. OSBPL9 is a novel ER stress-responsive gene and we confirmed that endogenous mRNA expression of OSBPL9 is upregulated by thapsigargin, and is repressed by IRE1α inhibitors, 4μ8C and toyocamycin, but not significantly by a PERK inhibitor, GSK2656157. These results demonstrate that this approach can be used to discover novel genes regulated by any stimuli without the need for microarray analysis, and that it can concomitantly produce reporter cells without identification of stimuli-responsive promoter/enhancer elements. Therefore, this system has a variety of benefits for basic and clinical research.

    DOI PubMed

  • An in vivo screening system to identify tumorigenic genes

    T. Ihara, Y. Hosokawa, K. Kumazawa, K. Ishikawa, J. Fujimoto, M. Yamamoto, T. Muramkami, N. Goshima, E. Ito, S. Watanabe, K. Semba

    ONCOGENE   36 ( 14 ) 2023 - 2029  2017.04  [Refereed]

     View Summary

    Screening for oncogenes has mostly been performed by in vitro transformation assays. However, some oncogenes might not exhibit their transforming activities in vitro unless putative essential factors from in vivo microenvironments are adequately supplied. Here, we have developed an in vivo screening system that evaluates the tumorigenicity of target genes. This system uses a retroviral high-efficiency gene transfer technique, a large collection of human cDNA clones corresponding to similar to 70% of human genes and a luciferase-expressing immortalized mouse mammary epithelial cell line (NMuMG-luc). From 845 genes that were highly expressed in human breast cancer cell lines, we focused on 205 genes encoding membrane proteins and/or kinases as that had the greater possibility of being oncogenes or drug targets. The 205 genes were divided into five subgroups, each containing 34-43 genes, and then introduced them into NMuMG-luc cells. These cells were subcutaneously injected into nude mice and monitored for tumor development by in vivo imaging. Tumors were observed in three subgroups. Using DNA microarray analyses and individual tumorigenic assays, we found that three genes, ADORA2B, PRKACB and LPAR3, were tumorigenic. ADORA2B and LPAR3 encode G-protein-coupled receptors and PRKACB encodes a protein kinase A catalytic subunit. Cells overexpressing ADORA2B, LPAR3 or PRKACB did not show transforming phenotypes in vitro, suggesting that transformation by these genes requires in vivo microenvironments. In addition, several clinical data sets, including one for breast cancer, showed that the expression of these genes correlated with lower overall survival rate.

    DOI

  • Hepatocyte nuclear factor 1 beta induces transformation and epithelial-to-mesenchymal transition

    Atsuka Matsui, Jiro Fujimoto, Kosuke Ishikawa, Emi Ito, Naoki Goshima, Shinya Watanabe, Kentaro Semba

    FEBS LETTERS   590 ( 8 ) 1211 - 1221  2016.04  [Refereed]

     View Summary

    Gene amplification can be a cause of cancer, and driver oncogenes have been often identified in amplified regions. However, comprehensive analysis of other genes coamplified with an oncogene is rarely performed. We focused on the 17q12-21 amplicon, which contains ERBB2. We established a screening system for oncogenic activity with the NMuMG epithelial cell line. We identified a homeobox gene, HNF1B, as a novel cooperative transforming gene. HNF1B induced cancerous phenotypes, which were enhanced by the coexpression of ERBB2, and induced epithelial-to-mesenchymal transition and invasive phenotypes. These results suggest that HNF1B is a novel oncogene that can work cooperatively with ERBB2.

    DOI

  • Enhanced expression of retinoic acid receptor alpha (RARA) induces epithelial-to-mesenchymal transition and disruption of mammary acinar structures

    Ayano Doi, Kosuke Ishikawa, Nao Shibata, Emi Ito, Jiro Fujimoto, Mizuki Yamamoto, Hatsuki Shiga, Hiromi Mochizuki, Yoshifumi Kaulamura, Naoki Goshima, Kentaro Semba, Shinya Watanabe

    MOLECULAR ONCOLOGY   9 ( 2 ) 355 - 364  2015.02  [Refereed]

     View Summary

    The early steps of mammary tumorigenesis include loss of epithelial cell polarity, escape from anoikis, and acquisition of proliferative capacity. The genes responsible for these processes are predicted to be early diagnostic markers or new therapeutic targets. Here we tested 51 genes coamplified with ERBB2 in the 17q12-21 amplicon for these tumorigenic activities using an MCF10A 3D culture-based screening system. We found that overexpression of retinoic acid receptor alpha (RARA) disrupted normal acinar structure and induced epithelial-to-mesenchymal transition (EMT). The mRNA levels of known EMT-inducing factors, including SLUG, FOXC2, ZEB1, and ZEB2, were significantly increased upon RARA overexpression. Knockdown of ZEB1 suppressed the RARA-mediated EMT phenotype. These results suggest that overexpression of RARA enhances malignant transformation during mammary tumorigenesis. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI

  • Gene amplification: mechanisms and involvement in cancer.

    Matsui, A, Ihara, T, Suda, H, Mikami, H, Semba, K

    Biomol Concepts   4   567 - 582  2013

  • Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene

    Makoto Saito, Yukiko Kato, Emi Ito, Jiro Fujimoto, Kosuke Ishikawa, Ayano Doi, Kentaro Kumazawa, Atsuka Matsui, Shiori Takebe, Takaomi Ishida, Sakura Azuma, Hiromi Mochizuki, Yoshifumi Kawamura, Yuka Yanagisawa, Reiko Honma, Jun-ichi Imai, Hirokazu Ohbayashi, Naoki Goshima, Kentaro Semba, Shinya Watanabe

    FEBS LETTERS   586 ( 12 ) 1708 - 1714  2012.06  [Refereed]

     View Summary

    Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers. (c) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI

  • NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells

    Noritaka Yamaguchi, Tetsunari Oyama, Emi Ito, Hitoshi Satoh, Sakura Azuma, Mitsuhiro Hayashi, Ken Shimizu, Reiko Honma, Yuka Yanagisawa, Akira Nishikawa, Mika Kawamura, Jun-Ichi Imai, Susumu Ohwada, Kuniaki Tatsuta, Jun-Ichiro Inoue, Kentaro Semba, Shinya Watanabe

    CANCER RESEARCH   68 ( 6 ) 1881 - 1888  2008.03  [Refereed]

     View Summary

    ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted. apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

    DOI

  • FoxA1 as a lineage-specific oncogene in luminal type breast cancer

    Noritaka Yamaguchi, Emi Ito, Sakura Azuma, Reiko Honma, Yuka Yanagisawa, Akira Nishikawa, Mika Kawamura, Jun-ichi Imai, Kuniaki Tatsuta, Jun-ichiro Inoue, Kentaro Semba, Shinya Watanabe

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   365 ( 4 ) 711 - 717  2008.01

     View Summary

    The forkhead transcription factor FoxA1 is thought to be involved in mammary tumorigenesis. However, the precise role of FoxA1 in breast cancer development is controversial. We examined expression of FoxA I in 35 human breast cancer cell lines and compared it with that of ErbB2, a marker of poor prognosis in breast cancer. We found that FoxA1 is expressed at high levels in all ErbB2-positive cell lines and a subset of ErbB2-negative cell lines. Down-regulation of FoxA1 by RNA interference significantly suppressed proliferation of ErbB2-negative and FoxA1-positive breast cancer cell lines. Down-regulation of FoxA1 also enhanced the toxic effect of Herceptin on ErbB2-positive cell lines through induction of apoptosis. Taken together with previous data that FoxA1 is a marker of luminal cells in mammary gland, our present results suggest that FoxA1 plays an important role as a lineage-specific oncogene in proliferation of cancer cells derived from mammary luminal cells. (c) 2007 Elsevier Inc. All rights reserved.

    DOI

  • Novel clusters of highly expressed genes accompany genomic amplification in breast cancers

    Emi Ito, Reiko Honma, Yuka Yanagisawa, Jun-Ichi Imai, Sakura Azuma, Tetsunari Oyama, Susumu Ohwada, Tetsu Akiyama, Nobuo Nomura, Jun-Ichiro Inoue, Shinya Watanabe, Kentaro Semba

    FEBS LETTERS   581 ( 21 ) 3909 - 3914  2007.08  [Refereed]

     View Summary

    Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI

  • Targeting c-Jun is a potential therapy of luminal breast cancer bone metastasis

    Yuxuan Han, Mitsuru Futakuchi, Kazuya Nakamichi, Yutaro Wakabayashi, Mai Sakamoto, Jun Nakayama, Kentaro Semba

       2022.07

     View Summary

    Luminal breast cancer has the highest bone metastasis frequency among all breast cancer subtypes, but its metastatic mechanism has not been elucidated because of the lack of appropriate metastatic cell lines. The study aim was to characterize high-osteolytic bone metastatic MCF7-BM cell lines and extract c-Jun, a novel bone metastasis marker. We found that c-Jun was upregulated in MCF7-BM cells, and its deficiency was associated with suppression of the cell migration, transformation, and stemness of BM cells. In vivo, c-Jun-deficient MCF7-TAM67 cells exhibited weaker bone metastatic ability. Additionally, c-Jun overexpression in MCF7-BM cells led to a tumor-migration promotion cycle in the bone microenvironment possibly by enhancing calcium-induced migration and releasing the osteoclast activator BMP5. Inhibition of c-Jun by JNK-IN-8, a JNK inhibitor, effectively reduced tumorigenesis activities and bone metastatic tumors. Our results indicate the potential benefits of a therapy that targets c-Jun to prevent or minimize luminal breast cancer bone metastasis.

    DOI

  • Metalloproteinase-Dependent and TMPRSS2-Independent Cell Surface Entry Pathway of SARS-CoV-2 Requires the Furin Cleavage Site and the S2 Domain of Spike Protein

    Mizuki Yamamoto, Jin Gohda, Ayako Kobayashi, Keiko Tomita, Youko Hirayama, Naohiko Koshikawa, Motoharu Seiki, Kentaro Semba, Tetsu Akiyama, Yasushi Kawaguchi, Jun-ichiro Inoue

    mBio    2022.06

     View Summary

    To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2. SARS-CoV-2 binds to the cell surface receptor ACE2 via the spike protein, and then the spike protein is cleaved by host proteases to enable entry.

    DOI

  • Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models.

    Yuxuan Han, Kazushi Azuma, Shinya Watanabe, Kentaro Semba, Jun Nakayama

    Clinical & experimental metastasis    2022.02  [Refereed]  [International journal]

     View Summary

    Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

    DOI PubMed

  • Epithelial cells remove precancerous cells by cell competition via MHC class I-LILRB3 interaction.

    Shiyu Ayukawa, Nagisa Kamoshita, Jun Nakayama, Ryohei Teramoto, Novalia Pishesha, Kenji Ohba, Nanami Sato, Kei Kozawa, Hikari Abe, Kentaro Semba, Nobuhito Goda, Yasuyuki Fujita, Takeshi Maruyama

    Nature immunology   22 ( 11 ) 1391 - 1402  2021.11  [Refereed]  [International journal]

     View Summary

    Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.

    DOI PubMed

  • Identification of two cancer stem cell-like populations in triple-negative breast cancer xenografts

    Jun Nakayama, Hiroko Matsunaga, Koji Arikawa, Takuya Yoda, Masahito Hosokawa, Haruko Takeyama, Yusuke Yamamoto, Kentaro Semba

       2021.10

     View Summary

    Abstract

    Gene expression analysis at the single-cell level by next generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell–cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-seq (scRNA-seq) datasets confirmed that the two CSC-like populations existed both in TNBC xenograft models and TNBC patients. In addition, the gene signature of the HMGA1-high CSC-like cluster has the potential to serve as a novel biomarker for diagnosis. The diversity of these multiple CSC-like populations may cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

    DOI

  • 溶骨性luminal乳がん細胞株の性状解析

    韓 宇軒, 中山 淳, 二口 充, 伊藤 恵美, 渡辺 慎哉, 仙波 憲太郎

    日本癌学会総会記事   80回   [E10 - 2]  2021.09

  • 同所性乳がん高転移株におけるNIKの機能解析

    林 祐介, 中山 淳, 山本 瑞生, 井上 純一郎, 山本 雄介, 仙波 憲太郎

    日本癌学会総会記事   80回   [J10 - 2]  2021.09

  • 乳がんにおいてアンドロゲン受容体は細胞老化を誘導する

    黒岩 由佳, 中山 淳, 仙波 憲太郎, 山本 雄介

    日本癌学会総会記事   80回   [P8 - 7]  2021.09

  • 高悪性度漿液性卵巣がんにおける受容体型チロシンキナーゼTIE1の機能解析

    松山 貴弥, 中山 淳, 渡辺 慎哉, 山本 雄介, 仙波 憲太郎

    日本癌学会総会記事   80回   [P10 - 4]  2021.09

  • Aberrant accumulation of NIK promotes tumorigenicity by dysregulating post-translational modifications in breast cancer

    Yusuke Hayashi, Jun Nakayama, Mizuki Yamamoto, Masashi Maekawa, Shinya Watanabe, Shigeki Higashiyama, Jun-ichiro Inoue, Yusuke Yamamoto, Kentaro Semba

       2021.08

     View Summary

    Post-translational modifications and mRNA translation are frequently altered in human cancers. However, investigations to understand their roles in the cancer progression mechanism remain insufficient. In this research, we explored protein levels altered by translational or post-translational regulation by analyzing transcriptome and western blotting data of the highly malignant breast cancer cell lines. From these analyses, NIK was found to be upregulated at the protein level to predominantly activate the non-canonical NF-κB pathway in a breast cancer cell line. Furthermore, the increase in NIK protein production was attributed to the dysregulation of ubiquitin-proteasome system caused by a decrease in the translation of cIAP1. NIK upregulation contributed to tumorigenicity by regulating the expression of inflammatory response-related genes. Collectively, our study suggests that NIK is post-translationally modified and has the potential to be a therapeutic target and diagnostic marker for breast cancer.

    DOI

  • HOXB7 induces STAT3-mediated transformation and lung metastasis in an immortalized mammary gland NMuMG cells

    Kazushi Azuma, Mai Sakamoto, Jun Nakayama, Shota Katayama, Atsuka Matsui, Naoki Goshima, Shinya Watanabe, Kentaro Semba

    bioRxiv    2021.05

     View Summary

    Abstract

    Genes from the homeobox family are often dysregulated in cancer. Particularly, homeobox B7 (HOXB7) amplification and overexpression correlate with poor prognosis in various cancers, such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, drug resistance, and angiogenesis. In this study, we show that HOXB7 overexpression correlates with a poor prognosis in patients with breast cancer. Coamplification of HOXB7 with ERBB2 enhanced the expression of ERBB2 and the malignancy of HER2-positive breast cancer cells. Additionally, HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line, NMuMG, induced cellular transformation through an activation of the JAK-STAT signaling pathway, which resulted in lung metastasis upon orthotropic transplantation in mice. These findings reveal that HOXB7 alone is sufficient to induce tumorigenesis and lung metastasis using HOXB7-expressing NMuMG cells.

    DOI

  • HOXB7 induces oncogenic transformation in NMuMG cells via JAK2-STAT3 signaling

    Mai Sakamoto, Jun Nakayama, Atsuka Matsui, Jiro Fujimoto, Naoki Goshima, Shinya Watanabe, Kentaro Semba

       2021.05

     View Summary

    The homeobox family genes are often dysregulated in various cancer types. In particular, HOXB7 overexpression contributes to cancer progression by promoting epithelial to mesenchymal transition, anti-cancer drug resistance, and metastasis of breast, hepatocellular and gastric cancer. Although the relationship between HOXB7 and cancer progression has been described, the role of HOXB7 in cancer initiation is unclear. In this study, we showed that HOXB7 overexpression induced oncogenic transformation in vitro and in vivo through the activation of JAK-STAT signaling and enhanced the expression of ERBB2 in NMuMG cells. In public data sets, HER2-positive breast cancer highly expressed HOXB7, the expression of which was correlated with poor prognosis in breast cancer cohorts. Furthermore, the amplification of HOXB7 on 17q23.32 was found to be a potential clinical diagnostic marker.

    DOI

  • The In Vivo Selection Method in Breast Cancer Metastasis.

    Jun Nakayama, Yuxuan Han, Yuka Kuroiwa, Kazushi Azuma, Yusuke Yamamoto, Kentaro Semba

    International journal of molecular sciences   22 ( 4 )  2021.02  [International journal]

     View Summary

    Metastasis is a complex event in cancer progression and causes most deaths from cancer. Repeated transplantation of metastatic cancer cells derived from transplanted murine organs can be used to select the population of highly metastatic cancer cells; this method is called as in vivo selection. The in vivo selection method and highly metastatic cancer cell lines have contributed to reveal the molecular mechanisms of cancer metastasis. Here, we present an overview of the methodology for the in vivo selection method. Recent comparative analysis of the transplantation methods for metastasis have revealed the divergence of metastasis gene signatures. Even cancer cells that metastasize to the same organ show various metastatic cascades and gene expression patterns by changing the transplantation method for the in vivo selection. These findings suggest that the selection of metastasis models for the study of metastasis gene signatures has the potential to influence research results. The study of novel gene signatures that are identified from novel highly metastatic cell lines and patient-derived xenografts (PDXs) will be helpful for understanding the novel mechanisms of metastasis.

    DOI PubMed

  • High Sensitivity In Vivo Imaging of Cancer Metastasis Using a Near-Infrared Luciferin Analogue seMpai.

    Jun Nakayama, Ryohei Saito, Yusuke Hayashi, Nobuo Kitada, Shota Tamaki, Yuxuan Han, Kentaro Semba, Shojiro A Maki

    International journal of molecular sciences   21 ( 21 )  2020.10  [Refereed]  [International journal]

     View Summary

    Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine.

    DOI PubMed

  • 同所性移植手法を用いた乳がん肺転移株のシグナル解析

    林 祐介, 中山 淳, 山本 瑞生, 山本 雄介, 井上 純一郎, 仙波 憲太郎

    日本癌学会総会記事   79回   OJ10 - 5  2020.10

  • 脳組織中における増殖能に基づくHER2陽性乳がん細胞株の分類

    黒岩 由佳, 中山 淳, 山本 雄介, 渡辺 慎哉, 仙波 憲太郎

    日本癌学会総会記事   79回   OJ10 - 6  2020.10

  • 同所性移植手法を用いた乳がん肺転移株のシグナル解析

    林 祐介, 中山 淳, 山本 瑞生, 山本 雄介, 井上 純一郎, 仙波 憲太郎

    日本癌学会総会記事   79回   OJ10 - 5  2020.10

  • 脳組織中における増殖能に基づくHER2陽性乳がん細胞株の分類

    黒岩 由佳, 中山 淳, 山本 雄介, 渡辺 慎哉, 仙波 憲太郎

    日本癌学会総会記事   79回   OJ10 - 6  2020.10

  • The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner.

    Mizuki Yamamoto, Maki Kiso, Yuko Sakai-Tagawa, Kiyoko Iwatsuki-Horimoto, Masaki Imai, Makoto Takeda, Noriko Kinoshita, Norio Ohmagari, Jin Gohda, Kentaro Semba, Zene Matsuda, Yasushi Kawaguchi, Yoshihiro Kawaoka, Jun-Ichiro Inoue

    Viruses   12 ( 6 )  2020.06  [Refereed]  [International journal]

     View Summary

    Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

    DOI PubMed

  • 頭蓋内移植手法によるHER2陽性乳がんの脳組織中における増殖能の評価

    黒岩 由佳, 中山 淳, 仙波 憲太郎

    日本細胞生物学会大会講演要旨集   72回   6 - 9  2020.06

  • 同所性移植手法を用いた乳がん肺転移株のシグナル解析

    林 祐介, 中山 淳, 仙波 憲太郎

    日本細胞生物学会大会講演要旨集   72回   6 - 26  2020.06

  • Structural analysis of TIFA: Insight into TIFA-dependent signal transduction in innate immunity.

    Teruya Nakamura, Chie Hashikawa, Kohtaro Okabe, Yuya Yokote, Mami Chirifu, Sachiko Toma-Fukai, Narushi Nakamura, Mihoko Matsuo, Miho Kamikariya, Yoshinari Okamoto, Jin Gohda, Taishin Akiyama, Kentaro Semba, Shinji Ikemizu, Masami Otsuka, Jun-Ichiro Inoue, Yuriko Yamagata

    Scientific reports   10 ( 1 ) 5152 - 5152  2020.03  [Refereed]  [International journal]

     View Summary

    TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-β-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.

    DOI PubMed

  • Generation of Rat Monoclonal Antibodies Specific for Human Stromal Cell-Derived Factor-2.

    Tanaka M, Shiota M, Koyama M, Nakayama J, Yashiro M, Semba K, Goda N

    Monoclonal antibodies in immunodiagnosis and immunotherapy   39 ( 1 ) 23 - 26  2020.02  [Refereed]

    DOI PubMed

  • Fibrosis growth factor 23 is a promoting factor for cardiac fibrosis in the presence of transforming growth factor-β1.

    Kazuhiro Kuga, Yoichiro Kusakari, Ken Uesugi, Kentaro Semba, Takashi Urashima, Toru Akaike, Susumu Minamisawa

    PloS one   15 ( 4 ) e0231905  2020  [Refereed]  [International journal]

     View Summary

    Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-β1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-β1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-β1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-β1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-β1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.

    DOI PubMed

  • 尾動脈注射手法を用いた溶骨性luminal乳がん細胞株の樹立と性状解析(Establishment and Characterization of Luminal Osteolytic Breast Cancer Cell Using Intra-Caudal Arterial Injection)

    韓 宇軒, 中山 淳, 二口 充, 伊藤 恵美, 渡辺 慎哉, 仙波 憲太郎

    日本癌学会総会記事   78回   P - 1274  2019.09

  • Cullin-3/KCTD10 complex is essential for K27-polyubiquitination of EIF3D in human hepatocellular carcinoma HepG2 cells.

    Maekawa M, Hiyoshi H, Nakayama J, Kido K, Sawasaki T, Semba K, Kubota E, Joh T, Higashiyama S

    Biochemical and biophysical research communications   516 ( 4 ) 1116 - 1122  2019.09  [Refereed]  [International journal]

     View Summary

    Eukaryotic translation initiation factor 3 subunit D (EIF3D) binds to the 5'-cap of specific mRNAs, initiating their translation into polypeptides. From a pathological standpoint, EIF3D has been observed to be essential for cell growth in various cancer types, and cancer patients with high EIF3D mRNA levels exhibit poor prognosis, indicating involvement of EIF3D in oncogenesis. In this study, we found, by mass spectrometry, that Cullin-3 (CUL3)/KCTD10 ubiquitin (Ub) ligase forms a complex with EIF3D. We also demonstrated that EIF3D is K27-polyubiquitinated at the lysine 153 and 275 residues in a KCTD10-dependent manner in human hepatocellular carcinoma HepG2 cells. Similar to other cancers, high expression of EIF3D significantly correlated with poor prognosis in hepatocellular carcinoma patients, and depletion of EIF3D drastically suppressed HepG2 cell proliferation. These results indicate that EIF3D is a novel substrate of CUL3/KCTD10 Ub ligase and suggest involvement of K27-polyubiquitinated EIF3D in the development of hepatocellular carcinoma.

    DOI PubMed

  • Cullin-3/KCTD10 E3 complex is essential for Rac1 activation through RhoB degradation in human epidermal growth factor receptor 2-positive breast cancer cells.

    Murakami A, Maekawa M, Kawai K, Nakayama J, Araki N, Semba K, Taguchi T, Kamei Y, Takada Y, Higashiyama S

    Cancer science   110 ( 2 ) 650 - 661  2019.02  [Refereed]  [International journal]

     View Summary

    Rho GTPase Rac1 is a central regulator of F-actin organization and signal transduction to control plasma membrane dynamics and cell proliferation. Dysregulated Rac1 activity is often observed in various cancers including breast cancer and is suggested to be critical for malignancy. Here, we showed that the ubiquitin E3 ligase complex Cullin-3 (CUL3)/KCTD10 is essential for epidermal growth factor (EGF)-induced/human epidermal growth factor receptor 2 (HER2)-dependent Rac1 activation in HER2-positive breast cancer cells. EGF-induced dorsal membrane ruffle formation and cell proliferation that depends on both Rac1 and HER2 were suppressed in CUL3- or KCTD10-depleted cells. Mechanistically, CUL3/KCTD10 ubiquitinated RhoB for degradation, another Rho GTPase that inhibits Rac1 activation at the plasma membrane by suppressing endosome-to-plasma membrane traffic of Rac1. In HER2-positive breast cancers, high expression of Rac1 mRNA significantly correlated with poor prognosis of the patients. This study shows that this novel molecular axis (CUL3/KCTD10/RhoB) positively regulates the activity of Rac1 in HER2-positive breast cancers, and our findings may lead to new treatment options for HER2- and Rac1-positive breast cancers.

    DOI PubMed

  • SNX9 determines the surface levels of integrin β1 in vascular endothelial cells: Implication in poor prognosis of human colorectal cancers overexpressing SNX9

    Kazufumi Tanigawa, Masashi Maekawa, Takeshi Kiyoi, Jun Nakayama, Riko Kitazawa, Sohei Kitazawa, Kentaro Semba, Tomohiko Taguchi, Satoshi Akita, Motohira Yoshida, Kei Ishimaru, Yuji Watanabe, Shigeki Higashiyama

    Journal of Cellular Physiology   234 ( 10 ) 17280 - 17294  2019.02  [Refereed]  [International journal]

     View Summary

    Angiogenesis, the formation of new blood vessels, is involved in a variety of diseases including the tumor growth. In response to various angiogenic stimulations, a number of proteins on the surface of vascular endothelial cells are activated to coordinate cell proliferation, migration, and spreading processes to form new blood vessels. Plasma membrane localization of these angiogenic proteins, which include vascular endothelial growth factor receptors and integrins, are warranted by intracellular membrane trafficking. Here, by using a siRNA library, we screened for the sorting nexin family that regulates intracellular trafficking and identified sorting nexin 9 (SNX9) as a novel angiogenic factor in human umbilical vein endothelial cells (HUVECs). SNX9 was essential for cell spreading on the Matrigel, and tube formation that mimics in vivo angiogenesis in HUVECs. SNX9 depletion significantly delayed the recycling of integrin β1, an essential adhesion molecule for angiogenesis, and reduced the surface levels of integrin β1 in HUVECs. Clinically, we showed that SNX9 protein was highly expressed in tumor endothelial cells of human colorectal cancer tissues. High-level expression of SNX9 messenger RNA significantly correlated with poor prognosis of the patients with colorectal cancer. These results suggest that SNX9 is an angiogenic factor and provide a novel target for the development of new antiangiogenic drugs.

    DOI PubMed

  • TRAF6 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell expansion.

    Mizuki Yamamoto, Chiho Abe, Sakura Wakinaga, Kota Sakane, Yo Yumiketa, Yuu Taguchi, Takayuki Matsumura, Kosuke Ishikawa, Jiro Fujimoto, Kentaro Semba, Maki Miyauchi, Taishin Akiyama, Jun-Ichiro Inoue

    Communications biology   2   292 - 292  2019  [Refereed]  [International journal]

     View Summary

    Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.

    DOI PubMed

  • PHLDA1, another PHLDA family protein that inhibits Akt.

    Yu Chen, Masahiro Takikawa, Shuichi Tsutsumi, Yoko Yamaguchi, Atsushi Okabe, Mayuna Shimada, Tatsuya Kawase, Akane Sada, Issei Ezawa, Yuhei Takano, Kisaburo Nagata, Yutaka Suzuki, Kentaro Semba, Hiroyuki Aburatani, Rieko Ohki

    Cancer science   109 ( 11 ) 3532 - 3542  2018.11  [Refereed]  [International journal]

     View Summary

    The PHLDA family (pleckstrin homology-like domain family) of genes consists of 3 members: PHLDA1, 2, and 3. Both PHLDA3 and PHLDA2 are phosphatidylinositol (PIP) binding proteins and function as repressors of Akt. They have tumor suppressive functions, mainly through Akt inhibition. Several reports suggest that PHLDA1 also has a tumor suppressive function; however, the precise molecular functions of PHLDA1 remain to be elucidated. Through a comprehensive screen for p53 target genes, we identified PHLDA1 as a novel p53 target, and we show that PHLDA1 has the ability to repress Akt in a manner similar to that of PHLDA3 and PHLDA2. PHLDA1 has a so-called split PH domain in which the PH domain is divided into an N-terminal (β sheets 1-3) and a C-terminal (β sheets 4-7 and an α-helix) portions. We show that the PH domain of PHLDA1 is responsible for its localization to the plasma membrane and binding to phosphatidylinositol. We also show that the function of the PH domain is essential for Akt repression. In addition, PHLDA1 expression analysis suggests that PHLDA1 has a tumor suppressive function in breast and ovarian cancers.

    DOI PubMed

  • Antitumor profile of the PI3K inhibitor ZSTK474 in human sarcoma cell lines.

    Nachi Namatame, Naomi Tamaki, Yuya Yoshizawa, Mutsumi Okamura, Yumiko Nishimura, Kanami Yamazaki, Miwa Tanaka, Takuro Nakamura, Kentaro Semba, Takao Yamori, Shin-Ichi Yaguchi, Shingo Dan

    Oncotarget   9 ( 80 ) 35141 - 35161  2018.10  [Refereed]  [International journal]

     View Summary

    Treatment of patients with advanced sarcoma remains challenging due to lack of effective medicine, with the development of novel drugs being of keen interest. A pan-PI3K inhibitor, ZSTK474, has been evaluated in clinical trials against a range of advanced solid tumors, with clinical benefit shown in sarcoma patients. In the present study, we developed a panel of 14 human sarcoma cell lines and investigated the antitumor effect of 24 anticancer agents including ZSTK474, other PI3K inhibitors, and those clinically used for sarcoma treatment. ZSTK474 exhibited a similar antiproliferative profile to other PI3K inhibitors but was clearly different from the other drugs examined. Indeed, ZSTK474 inhibited PI3K-downstream pathways, in parallel to growth inhibition, in all cell lines examined, showing proof-of-concept of PI3K inhibition. In addition, ZSTK474 induced apoptosis selectively in Ewing's sarcoma (RD-ES and A673), alveolar rhabdomyosarcoma (SJCRH30) and synovial sarcoma (SYO-1, Aska-SS and Yamato-SS) cell lines, all of which harbor chromosomal translocation and resulting oncogenic fusion genes, EWSR1-FLI1, PAX3-FOXO1 and SS18-SSX, respectively. Finally, animal experiments confirmed the antitumor activity of ZSTK474 in vivo, with superior efficacy observed in translocation-positive cells. These results suggest that ZSTK474 could be a promising drug candidate for treating sarcomas, especially those harboring chromosomal translocation.

    DOI PubMed

  • 腫瘍内微小不均一性解明のための空間トランスクリプトミクス解析技術の確立(Establishment of Spatial Transcriptomics for Analysis of Tumor Microenvironment and Heterogeneity)

    中山 淳, 仙波 憲太郎

    日本癌学会総会記事   77回   1594 - 1594  2018.09

  • Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells

    Tatsuyuki Matsudaira, Kojiro Mukai, Taishin Noguchi, Junya Hasegawa, Tomohisa Hatta, Shun-ichiro Iemura, Tohru Natsume, Norio Miyamura, Hiroshi Nishina, Jun Nakayama, Kentaro Semba, Takuya Tomita, Shigeo Murata, Hiroyuki Arai, Tomohiko Taguchi

    NATURE COMMUNICATIONS   8   1246  2017.11  [Refereed]

     View Summary

    Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.

    DOI

  • Intratumoral bidirectional transitions between epithelial and mesenchymal cells in triple-negative breast cancer.

    Mizuki Yamamoto, Kota Sakane, Kana Tominaga, Noriko Gotoh, Takayoshi Niwa, Yasuko Kikuchi, Keiichiro Tada, Naoki Goshima, Kentaro Semba, Jun-Ichiro Inoue

    Cancer science   108 ( 6 ) 1210 - 1222  2017.06  [Refereed]  [International journal]

     View Summary

    Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition MET, are crucial in several stages of cancer metastasis. Epithelial-mesenchymal transition allows cancer cells to move to proximal blood vessels for intravasation. However, because EMT and MET processes are dynamic, mesenchymal cancer cells are likely to undergo MET transiently and subsequently re-undergo EMT to restart the metastatic process. Therefore, spatiotemporally coordinated mutual regulation between EMT and MET could occur during metastasis. To elucidate such regulation, we chose HCC38, a human triple-negative breast cancer cell line, because HCC38 is composed of epithelial and mesenchymal populations at a fixed ratio even though mesenchymal cells proliferate significantly more slowly than epithelial cells. We purified epithelial and mesenchymal cells from Venus-labeled and unlabeled HCC38 cells and mixed them at various ratios to follow EMT and MET. Using this system, we found that the efficiency of EMT is approximately an order of magnitude higher than that of MET and that the two populations significantly enhance the transition of cells from the other population to their own. In addition, knockdown of Zinc finger E-box-binding homeobox 1 (ZEB1) or Zinc finger protein SNAI2 (SLUG) significantly suppressed EMT but promoted partial MET, indicating that ZEB1 and SLUG are crucial to EMT and MET. We also show that primary breast cancer cells underwent EMT that correlated with changes in expression profiles of genes determining EMT status and breast cancer subtype. These changes were very similar to those observed in EMT in HCC38 cells. Consequently, we propose HCC38 as a suitable model to analyze EMT-MET dynamics that could affect the development of triple-negative breast cancer.

    DOI PubMed

  • The transcription factor MAFK induces EMT and malignant progression of triple-negative breast cancer cells through its target GPNMB.

    Yukari Okita, Minori Kimura, Rudy Xie, Chen Chen, Larina Tzu-Wei Shen, Yurika Kojima, Hiroyuki Suzuki, Masafumi Muratani, Masao Saitoh, Kentaro Semba, Carl-Henrik Heldin, Mitsuyasu Kato

    Science signaling   10 ( 474 )  2017.04  [International journal]

     View Summary

    Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.

    DOI PubMed

  • Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells

    Tianshu Li, Takuya Amari, Kentaro Semba, Tadashi Yamamoto, Shinji Takeoka

    NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE   13 ( 3 ) 1219 - 1227  2017.04  [Refereed]

     View Summary

    1,5-Dihexadecyl N, N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLGliposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 mu g/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Type I neuregulin1 alpha is a novel local mediator to suppress hepatic gluconeogenesis in mice

    Takatomo Arai, Yumika Ono, Yujiro Arimura, Keimon Sayama, Tomohiro Suzuki, Satoko Shinjo, Mai Kanai, Shin-ichi Abe, Kentaro Semba, Nobuhito Goda

    SCIENTIFIC REPORTS   7   42959  2017.02  [Refereed]

     View Summary

    Neuregulin1 is an epidermal growth factor (EGF)-like domain-containing protein that has multiple isoforms and functions as a local mediator in the control of various cellular functions. Here we show that type I isoform of neuregulin1 with an alpha-type EGF-like domain (Nrg1 alpha) is the major isoform in mouse liver and regulates hepatic glucose production. Forced expression of Nrg1 alpha in mouse liver enhanced systemic glucose disposal and decreased hepatic glucose production with reduced fasting blood glucose levels. Nuclear forkhead box protein O1 (FoxO1) and its downstream targets, PEPCK and G6Pase, were suppressed in liver and isolated hepatocytes by Nrg1 alpha overexpression. In contrast, silencing of Nrg1 alpha enhanced glucose production with increased PEPCK and G6Pase expressions in cAMP/dexamethasone-stimulated hepatocytes. Mechanistically, the recombinant alpha-type EGF-like domain of NRG1 alpha (rNRG1 alpha) stimulated the ERBB3 signalling pathway in hepatocytes, resulting in decreased nuclear FoxO1 accumulation via activation of both the AKT and ERK pathways. In addition, acute treatment with rNRG1 alpha also suppressed elevation of blood glucose levels after both glucose and pyruvate challenge. Although a liver-specific deletion of Nrg1 gene in mice showed little effect on systemic glucose metabolism, these results suggest that NRG1 alpha have a novel regulatory function in hepatic gluconeogenesis by regulating the ERBB3-AKT/ERK-FoxO1 cascade.

    DOI

  • Comparative analysis of gene regulatory networks of highly metastatic breast cancer cells established by orthotopic transplantation and intra-circulation injection

    Jun Nakayama, Emi Ito, Jiro Fujimoto, Shinya Watanabe, Kentaro Semba

    INTERNATIONAL JOURNAL OF ONCOLOGY   50 ( 2 ) 497 - 504  2017.02  [Refereed]

     View Summary

    Metastasis signature genes in breast cancer have been studied comparing transcriptomic profiles of highly metastatic cancer cell lines established by intra-circulation injection with that of their parental cell line. However, this method is not suitable to analyze the initial steps of metastasis including invasion into local tissues and the circulatory system. To characterize the molecular mechanisms of early metastasis, we established highly metastatic MDA-MB-231 cell lines that metastasized to lung by the two animal transplantation models: the orthotopic transplantation method, which mimics all steps of metastasis, or intra-circulation injection method. We then performed data-mining and network analysis of gene expression profiles of metastatic cell lines established by each transplantation method. Transcriptome analysis of seven metastatic cell lines revealed novel lung metastasis signature genes, including known metastasis promoting genes and signature genes. In the OXconc (orthotopic xenograft concentration) signature, 'chemotaxis' and 'cell adhesion' terms were enriched. In the TVIconc (tail vein injection concentration) signature, 'antigen recognition' and 'cell adhesion' were enriched. Furthermore, network analysis of the metastasis signature genes highlighted hub genes in the gene regulatory network. Our findings show that expression profiles of highly metastatic cell lines were different between the orthotopic transplantation and intra-circulation injection method. It also indicates that some metastatic signature genes have been missed in previous studies. Characterization of metastasis genes using the orthotopic transplantation method will be helpful in understanding the multi-step mechanisms of metastasis. Signature genes in OXconc may have the potential to become prognostic markers.

    DOI

  • Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation

    Hirotaka Hoshi, Gen Hiyama, Kosuke Ishikawa, Kiyoshi Inageda, Jiro Fujimoto, Ai Wakamatsu, Takushi Togashi, Yoshifumi Kawamura, Nobuhiko Takahashi, Arisa Higa, Naoki Goshima, Kentaro Semba, Shinya Watanabe, Motoki Takagi

    ONCOLOGY REPORTS   37 ( 1 ) 66 - 76  2017.01  [Refereed]  [International journal]

     View Summary

    Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell-based assay for evaluating the efficacy of anti-EGFR drugs against EGFR mutation.

    DOI PubMed

  • Novel p53 target gene FUCA1 encodes a fucosidase and regulates growth and survival of cancer cells

    Issei Ezawa, Yuichiro Sawai, Tatsuya Kawase, Atsushi Okabe, Shuichi Tsutsumi, Hitoshi Ichikawa, Yuka Kobayashi, Fumio Tashiro, Hideo Namiki, Tadashi Kondo, Kentaro Semba, Hiroyuki Aburatani, Yoichi Taya, Hitoshi Nakagama, Rieko Ohki

    CANCER SCIENCE   107 ( 6 ) 734 - 745  2016.06  [Refereed]

     View Summary

    The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and ChIP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, -l-1 (FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression.

    DOI

  • IER5 generates a novel hypophosphorylated active form of HSF1 and contributes to tumorigenesis

    Yoshinori Asano, Tatsuya Kawase, Atsushi Okabe, Shuichi Tsutsumi, Hitoshi Ichikawa, Satoko Tatebe, Issay Kitabayashi, Fumio Tashiro, Hideo Namiki, Tadashi Kondo, Kentaro Semba, Hiroyuki Aburatani, Yoichi Taya, Hitoshi Nakagama, Rieko Ohki

    SCIENTIFIC REPORTS   6   19174  2016.01  [Refereed]

     View Summary

    The transcription factors HSF1 and p53 both modulate the stress response, thereby protecting and facilitating the recovery of stressed cells, but both have the potential to promote tumor development. Here we show that a p53 target gene, IER5, encodes an activator of HSF1. IER5 forms a ternary complex with HSF1 and the phosphatase PP2A, and promotes the dephosphorylation of HSF1 at numbers of serine and threonine residues, generating a novel, hypo-phosphorylated active form of HSF1. IER5 is also transcriptionally upregulated in various cancers, although this upregulation is not always p53dependent. The IER5 locus is associated with a so-called super enhancer, frequently associated with hyperactivated oncogenes in cancer cell lines. Enhanced expression of IER5 induces abnormal HSF1 activation in cancer cells and contributes to the proliferation of these cells under stressed conditions. These results reveal the existence of a novel IER5-mediated cancer regulation pathway that is responsible for the activation of HSF1 observed in various cancers.

    DOI

  • The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia

    Hajime Yamauchi, Takanori Matsumaru, Tomoko Morita, Susumu Ishikawa, Katsumi Maenaka, Ichigaku Takigawa, Kentaro Semba, Shunsuke Kon, Yasuyuki Fujita

    SCIENTIFIC REPORTS   5  2015.10

     View Summary

    Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.

    DOI

  • Tropomodulin 1 Expression Driven by NF-kappa B Enhances Breast Cancer Growth

    Taku Ito-Kureha, Naohiko Koshikawa, Mizuki Yamamoto, Kentaro Semba, Noritaka Yamaguchi, Tadashi Yamamoto, Motoharu Seiki, Jun-ichiro Inoue

    CANCER RESEARCH   75 ( 1 ) 62 - 72  2015.01  [Refereed]

     View Summary

    Triple-negative breast cancers (TNBC), which include the basallike and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-kappa B activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-kappa B target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-kappa B-inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-kappa B and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of beta-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-kappa B activation and MMP13 induction, which accounts in part for the NF-kappa B-dependent malignant phenotype of TNBC. (C)2014 AACR.

    DOI

  • Cell growth control by stable Rbg2/Gir2 complex formation under amino acid starvation

    Kosuke Ishikawa, Koichi Ito, Jun-ichiro Inoue, Kentaro Semba

    GENES TO CELLS   18 ( 10 ) 859 - 872  2013.10  [Refereed]

     View Summary

    The molecular fine-tuning mechanisms underlying adaptive responses to environmental stresses in eukaryotes remain largely unknown. Here, we report on a novel stress-induced cell growth control mechanism involving a highly conserved complex containing Rbg2 and Gir2 subunits, which are the budding yeast orthologs of human Drg2 and Dfrp2, respectively. We found that the complex is responsible for efficient cell growth under amino acid starvation. Using native PAGE analyses, we observed that, individually, Rbg2 and Gir2 were labile proteins. However, they formed a complex that stabilized each other, and this stability became significantly enhanced after amino acid starvation. We observed that the stabilization of the complex was strictly dependent on GDP or GTP binding to Rbg2. A point mutation (S77N) that inactivated nucleotide binding impaired formation of the complex and disrupted the stress-induced cell growth. Interestingly, the complex bound the translational activator Gcn1 in a dose-dependent manner according to the stress level, suggesting a dynamic association with the cellular translational machinery. We propose that the Rbg2/Gir2 complex is a modulator that maintains cellular homoeostasis, thus promoting the survival of eukaryotic organisms in stressful environments.

    DOI

  • Identification of integrin α3 as a molecular marker of cells undergoing epithelial-mesenchymal transition and of cancer cells with aggressive phenotypes.

    Takuya Shirakihara, Tomonori Kawasaki, Akihiko Fukagawa, Kentaro Semba, Ryuichi Sakai, Kohei Miyazono, Keiji Miyazawa, Masao Saitoh

    Cancer science   104 ( 9 ) 1189 - 97  2013.09  [Refereed]  [International journal]

     View Summary

    Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Transforming growth factor (TGF)-β induces EMT in mouse epithelial cells. During prolonged treatment, TGF-β successively induces myofibroblastic differentiation with increased expression of myofibroblast marker proteins, including smooth muscle α actin and calponin. We recently showed that fibroblast growth factor-2 prevented myofibroblastic differentiation induced by TGF-β, and transdifferentiated the cells to those with much more aggressive characteristics (enhanced EMT). To identify the molecular markers specifically expressed in cells undergoing enhanced EMT induced by the combination of TGF-β and fibroblast growth factor-2, we carried out a microarray-based analysis and found that integrin α3 (ITGA3) and Ret were upregulated. Intriguingly, ITGA3 was also overexpressed in breast cancer cells with aggressive phenotypes and its expression was correlated with that of δEF-1, a key regulator of EMT. Moreover, the expression of both genes was downregulated by U0126, a MEK 1/2 inhibitor. Therefore, ITGA3 is a potential marker protein for cells undergoing enhanced EMT and for cancer cells with aggressive phenotypes, which is positively regulated by δEF-1 and the MEK-ERK pathway.

    DOI PubMed

  • NF-kappa B non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

    Mizuki Yamamoto, Yuu Taguchi, Taku Ito-Kureha, Kentaro Semba, Noritaka Yamaguchi, Jun-ichiro Inoue

    NATURE COMMUNICATIONS   4   2299  2013.08  [Refereed]

     View Summary

    Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype, a subgroup of triple-negative breast cancer, exhibits high levels of constitutively active NF-kappa B signalling. Here we show that NF-kappa B activation, induced by inflammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-kappa B-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-kappa B has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.

    DOI

  • Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor

    S. Yoshikawa, M. Kukimoto-Niino, L. Parker, N. Handa, T. Terada, T. Fujimoto, Y. Terazawa, M. Wakiyama, M. Sato, S. Sano, T. Kobayashi, T. Tanaka, L. Chen, Z-J Liu, B-C Wang, M. Shirouzu, S. Kawa, K. Semba, T. Yamamoto, S. Yokoyama

    ONCOGENE   32 ( 1 ) 27 - 38  2013.01  [Refereed]

     View Summary

    The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations. Oncogene (2013) 32, 27-38; doi:10.1038/onc.2012.21; published online 20 February 2012

    DOI

  • Potential anti-tumor effect of IFN-lambda 2 (IL-28A) against human lung cancer cells

    Yasuhiro Tezuka, Shunsuke Endo, Aya Matsui, Atsuko Sato, Katsuyo Saito, Kentaro Semba, Masafumi Takahashi, Takashi Murakami

    LUNG CANCER   78 ( 3 ) 185 - 192  2012.12  [Refereed]

     View Summary

    Interferon (IFN)-lambda s (IL-28A/IL-28B/IL-29) classified as type III IFNs, are the latest members identified of the interferon family. As with type I IFNs such as IFN-alpha, type III IFNs share antiviral and antitumor activity and may have fewer side effects due to a more selective receptor distribution. Therefore, type III IFNs may be clinically useful for human viral and malignant diseases. Here we demonstrate the potential anti-tumor effect of IFN-lambda 2 (IL-28A) against human lung cancer cells. All lung cancer cell lines that we examined expressed both IFN-lambda receptors (IL-28R1 and IL-10R2). Lung cancer cells with epidermal growth factor receptor (EGFR) mutations were more sensitive to IFN-lambda 2 treatment compared with cells with KRAS mutations. HCC827 cells with an EGFR mutation treated with IFN-lambda 2 underwent growth suppression and apoptotic cell death by STAT1 phosphorylation. Administration of neutralizing antibodies to IFN-lambda inhibited caspase-3/7 activity induced by IFN-lambda 2. Finally, in vivo luminescent imaging also demonstrated the anti-tumor effect of IFN-lambda 2 in a cancer cell transplant animal model. Taken together, IFN-lambda 2 would be a new therapeutic agent for clinical lung cancers with EGFR mutations. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

    DOI

  • CXCL17 Expression by Tumor Cells Recruits CD11b(+)Gr1(high)F4/80(-) Cells and Promotes Tumor Progression

    Aya Matsui, Hideaki Yokoo, Yoichi Negishi, Yoko Endo-Takahashi, Nicole A. L. Chun, Ichiro Kadouchi, Ryo Suzuki, Kazuo Maruyama, Yukihiko Aramaki, Kentaro Semba, Eiji Kobayashi, Masafumi Takahashi, Takashi Murakami

    PLOS ONE   7 ( 8 ) e44080  2012.08  [Refereed]

     View Summary

    Background: Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression.
    Methodology/Principal Findings: CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b(+)Gr1(+) myeloid-derived cells at tumor sites in mice and promoted CD31(+) tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b(+)Gr1(high)F4/80(-) cells (similar to 90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b(+)Gr1(+) cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation.
    Conclusions/Significance: These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

    DOI

  • TGF-beta drives epithelial-mesenchymal transition through delta EF1-mediated downregulation of ESRP

    K. Horiguchi, K. Sakamoto, D. Koinuma, K. Semba, A. Inoue, S. Inoue, H. Fujii, A. Yamaguchi, K. Miyazawa, K. Miyazono, M. Saitoh

    ONCOGENE   31 ( 26 ) 3190 - 3201  2012.06  [Refereed]

     View Summary

    Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair and cancer progression in adult tissues. We have recently shown that transforming growth factor (TGF)-beta-induced EMT involves isoform switching of fibroblast growth factor receptors by alternative splicing. We performed a microarray-based analysis at single exon level to elucidate changes in splicing variants generated during TGF-beta-induced EMT, and found that TGF-beta induces broad alteration of splicing patterns by downregulating epithelial splicing regulatory proteins (ESRPs). This was achieved by TGF-beta-mediated upregulation of delta EF1 family proteins, delta EF1 and SIP1. delta EF1 and SIP1 each remarkably repressed ESRP2 transcription through binding to the ESRP2 promoter in NMuMG cells. Silencing of both delta EF1 and SIP1, but not either alone, abolished the TGF-beta-induced ESRP repression. The expression profiles of ESRPs were inversely related to those of delta EF1 and SIP in human breast cancer cell lines and primary tumor specimens. Further, overexpression of ESRPs in TGF-beta-treated cells resulted in restoration of the epithelial splicing profiles as well as attenuation of certain phenotypes of EMT. Therefore, delta EF1 family proteins repress the expression of ESRPs to regulate alternative splicing during TGF-beta-induced EMT and the progression of breast cancers. Oncogene (2012) 31, 3190-3201; doi:10.1038/onc.2011.493; published online 31 October 2011

    DOI

  • ErbB2/HER2: Its Contribution to Basic Cancer Biology and the Development of Molecular Targeted Therapy

    Tadashi Yamamoto, Makoto Saito, Kentaro Kumazawa, Ayano Doi, Atsuka Matsui, Shiori Takebe, Takuya Amari, Masaaki Oyama, Kentaro Semba

    Breast Cancer - Carcinogenesis, Cell Growth and Signalling Pathways    2011.11

    DOI

  • Epigenetic alteration of the NF-kappa B-inducing kinase (NIK) gene is involved in enhanced NIK expression in basal-like breast cancer

    Mizuki Yamamoto, Taku Ito, Takafumi Shimizu, Takaomi Ishida, Kentaro Semba, Shinya Watanabe, Noritaka Yamaguchi, Jun-ichiro Inoue

    CANCER SCIENCE   101 ( 11 ) 2391 - 2397  2010.11  [Refereed]

     View Summary

    Basal-like breast cancers are triple-negative (estrogen receptor negative, progesterone receptor negative, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) negative) tumors with an aggressive clinical behavior that lacks effective molecular targets for therapy. We reported previously that the basal-like subtype cell lines display high constitutive nuclear factor (NF)-kappa B activation, whose inhibition in the basal-like subtypes suppressed their proliferation. Moreover, NF-kappa B-inducing kinase (NIK) is involved in the constitutive NF-kappa B activation. Here, we report that enhanced NIK expression, which is exclusively observed in the basal-like subtype rather than the luminal-like subtype or non-tumorigenic mammary epithelial cells, is caused by epigenetic alteration of the NIK gene. The stability of NIK mRNA and transcriptional activity driven by the NIK promoter are similar in the basal-like and luminal-like subtypes. However, histone H3 acetylation levels were up-regulated in the basal-like subtype. Furthermore, treatment of the luminal-like subtype with a histone deacetylase inhibitor, valproic acid, significantly increased NIK expression. Although DNA methylation of the NIK locus was not detected, NIK expression also increased when the luminal-like subtype was treated with 5-azacytidine, which inhibits histone H3-Lys-9 dimethylation in addition to DNA methylation. Taken together, these results suggest that the closed chromatin structure mediated by histone H3 methylation and deacetylation suppresses NIK expression in the luminal-like subtype, whereas disruption of these suppression mechanisms leads to enhanced NIK expression and the constitutive NF-kappa B activation in the basal-like subtype. Thus, NIK and genes induced by the NIK-mediated constitutive NF-kappa B activation could be therapeutic targets of basal-like breast cancer. (Cancer Sci 2010; 101: 2391-2397).

    DOI

  • Phosphoproteomics-Based Modeling Defines the Regulatory Mechanism Underlying Aberrant EGFR Signaling

    Shinya Tasaki, Masao Nagasaki, Hiroko Kozuka-Hata, Kentaro Semba, Noriko Gotoh, Seisuke Hattori, Jun-ichiro Inoue, Tadashi Yamamoto, Satoru Miyano, Sumio Sugano, Masaaki Oyama

    PLOS ONE   5 ( 11 ) e13926  2010.11  [Refereed]

     View Summary

    Background: Mutation of the epidermal growth factor receptor (EGFR) results in a discordant cell signaling, leading to the development of various diseases. However, the mechanism underlying the alteration of downstream signaling due to such mutation has not yet been completely understood at the system level. Here, we report a phosphoproteomics-based methodology for characterizing the regulatory mechanism underlying aberrant EGFR signaling using computational network modeling.
    Methodology/Principal Findings: Our phosphoproteomic analysis of the mutation at tyrosine 992 (Y992), one of the multifunctional docking sites of EGFR, revealed network-wide effects of the mutation on EGF signaling in a time-resolved manner. Computational modeling based on the temporal activation profiles enabled us to not only rediscover already-known protein interactions with Y992 and internalization property of mutated EGFR but also further gain model-driven insights into the effect of cellular content and the regulation of EGFR degradation. Our kinetic model also suggested critical reactions facilitating the reconstruction of the diverse effects of the mutation on phosphoproteome dynamics.
    Conclusions/Significance: Our integrative approach provided a mechanistic description of the disorders of mutated EGFR signaling networks, which could facilitate the development of a systematic strategy toward controlling disease-related cell signaling.

    DOI

  • Identification of BCAP-(L) as a negative regulator of the TLR signaling-induced production of IL-6 and IL-10 in macrophages by tyrosine phosphoproteomics

    Takayuki Matsumura, Masaaki Oyama, Hiroko Kozuka-Hata, Kosuke Ishikawa, Takafumi Inoue, Tatsushi Muta, Kentaro Semba, Jun-ichiro Inoue

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   400 ( 2 ) 265 - 270  2010.09  [Refereed]

     View Summary

    Toll-like receptor (TLR) signaling in macrophages is essential for anti-pathogen responses such as cytokine production and antigen presentation. Although numerous reports suggest that protein tyrosine kinases (PTKs) are involved in cytokine induction in response to lipopolysaccharides (LPS; TLR4 ligand) in macrophages, the PTK-mediated signal transduction pathway has yet to be analyzed in detail. Here. we carried out a comprehensive and quantitative dynamic tyrosine phosphoproteomic analysis on the TLR4-mediated host defense system in RAW264.7 macrophages using stable isotope labeling by amino acids in cell culture (SILAC). We determined the temporal profiles of 25 proteins based on SILAC-encoded peptide(s). Of these, we focused on the tyrosine phosphorylation of B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) because the function of BCAP remains unknown in TLR signaling in macrophages. Furthermore, Bcap has two distinct transcripts, a full-length (Bcap-(L)) and an alternatively initiated or spliced (Bcap-(S)) mRNA, and little is known about the differential functions of the BCAP-L and BCAP-(S) proteins. Our study showed, for the first time, that RNAi-mediated selective depletion of BCAP-(L) enhanced IL-6 and IL-10 production but not TNF-alpha production in TLR ligand-stimulated macrophages. We propose that BCAP-(L) (but not BCAP-(S)) is a negative regulator of the TLR-mediated host defense system in macrophages. (C) 2010 Elsevier Inc. All rights reserved.

    DOI

  • Independent stabilizations of polysomal Drg1/Dfrp1 complex and non-polysomal Drg2/Dfrp2 complex in mammalian cells

    Kosuke Ishikawa, Taishin Akiyama, Koichi Ito, Kentaro Semba, Jun-ichiro Inoue

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   390 ( 3 ) 552 - 556  2009.12  [Refereed]

     View Summary

    Various widely known GTPases are associated with diverse crucial cellular processes. However, the functional targets of the universally conserved homologous GTPases Drg1 and Drg2, constituting the DRG subfamily in eukaryotes, remain completely unknown despite their pleiotropic cell growth effects. Contrary to expectations of functional redundancy between Drg1 and Drg2 due to their high homology, the different binding proteins Dfrp1 and Dfrp2, respectively, have been previously identified.
    Here, we report the first systematic characterization of all these proteins in mammals by analyses in physiological conditions. Our findings are: (11) At least one of the components of the Drg1/Dfrp1 and the Drg2/Dfrp2 complexes is specifically and drastically stabilized by each unique complex formation: and (2) the Drg1/Dfrp1 complex cosediments with polysome, while neither Drg2 nor Dfrp2 is found in ribosomal fractions at all.
    These results suggest that the Drg1/Dfrp1 complex independently modulates a protein synthesis mechanism different from the Drg2/Dfrp2 complex in mammalian cells. (C) 2009 Elsevier Inc. All rights reserved.

    DOI

  • NIK is involved in constitutive activation of the alternative NF-kappa B pathway and proliferation of pancreatic cancer cells

    Takashi Nishina, Noritaka Yamaguchi, Jin Gohda, Kentaro Semba, Jun-ichiro Inoue

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   388 ( 1 ) 96 - 101  2009.10  [Refereed]

     View Summary

    Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-kappa B is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-kappa B activation. Here, we show that the alternative pathway is constitutively activated and NF-kappa B-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer. (C) 2009 Elsevier Inc. All rights reserved.

    DOI

  • TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is a negative regulator of the TRAF6-induced cellular functions.

    Takayuki Matsumura, Junko Kawamura-Tsuzuku, Tadashi Yamamoto, Kentaro Semba, Jun-Ichiro Inoue

    Journal of biochemistry   146 ( 3 ) 375 - 81  2009.09  [International journal]

     View Summary

    Tumour necrosis factor receptor-associated factor (TRAF)-interacting protein with a forkhead-associated domain (TIFA) activates TRAF6 to induce NF-kappaB activation. TIFA-related protein, TIFAB, is highly expressed in the spleen and inhibits TIFA-mediated TRAF6 activation. However, little is known about cell types that express TIFAB and its function in those cells. Here, we show that TIFAB is mainly expressed in B cells rather than T cells in the spleen and that the expression level was much higher in dendritic cells (DCs) and macrophages than that in splenic lymphocytes. TIFAB expression was downregulated when B cells, DCs or macrophages were stimulated by TRAF6-mediated proliferative or maturation signals including those emanating from CD40, sIgM and TLRs. Furthermore, microinjection experiments using NIH3T3 cells revealed that TIFAB inhibited entry into the S phase of the cell cycle. Our results suggest that TIFAB could act as a negative regulator of the TRAF6-induced cellular function such as B cell proliferation and maturation of DCs and macrophages.

    DOI PubMed

  • Constitutive activation of nuclear factor-kappa B is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines

    Noritaka Yamaguchi, Taku Ito, Sakura Azuma, Emi Ito, Reiko Honma, Yuka Yanagisawa, Akira Nishikawa, Mika Kawamura, Jun-ichi Imai, Shinya Watanabe, Kentaro Semba, Jun-ichiro Inoue

    CANCER SCIENCE   100 ( 9 ) 1668 - 1674  2009.09  [Refereed]

     View Summary

    Constitutive nuclear factor (NF)-kappa B activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-kappa B activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-kappa B activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-kappa B activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-kappa B activation by expression of I kappa B alpha super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-kappa B-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-kappa B activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-kappa B activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. (Cancer Sci 2009; 100: 1668-1674).

    DOI

  • A novel mouse monoclonal antibody targeting ErbB2 suppresses breast cancer growth

    Seiji Kawa, Hirohisa Matsushita, Hirokazu Ohbayashi, Kentaro Semba, Tadashi Yamamoto

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   384 ( 3 ) 329 - 333  2009.07  [Refereed]

     View Summary

    Overexpression of ErbB2 in breast cancer is associated with increased recurrence and worse prognosis. Accumulating evidences suggest that molecular targeted therapy is a promising anticancer strategy. In this study, we produced a novel anti-ErB2 monoclonal antibody, 6G10, that recognized an epitope distinct from the trastuzumab binding site. 6G10 induced aggregation of BT474 breast cancer cells and inhibited proliferation of various breast cancer cell lines including BT474. A growth inhibition assay showed that 6G10 had EC(50) values comparable to trastuzumab, indicating that the drugs have a similar level of potency. Furthermore, intraperitoneal administration of 6G10 completely inhibited the growth of xenografted tumors derived from BT474 and SK-BR-3 cells. These data suggested that 6G10 has great therapeutic potential and could be administered to patients alternatively, or synergistically, with trastuzumab. (C) Elsevier Inc. All rights reserved.

    DOI

  • TRAF6 establishes innate immune responses by activating NF-κB and IRF7 upon sensing cytosolic viral RNA and DNA

    Hiroyasu Konno, Takuya Yamamoto, Kohsuke Yamazaki, Jin Gohda, Taishin Akiyama, Kentaro Semba, Hideo Goto, Atsushi Kato, Toshiaki Yujiri, Takahiko Imai, Yasushi Kawaguchi, Bing Su, Osamu Takeuchi, Shizuo Akira, Yasuko Tsunetsugu-Yokota, Jun Ichiro Inoue

    PLoS ONE   4 ( 5 ) e5674  2009.05  [Refereed]

     View Summary

    Background: In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. Principal Findings: Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7. Conclusions/Significance: Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection. © 2009 Konno et al.

    DOI PubMed

  • Temporal Perturbation of Tyrosine Phosphoproteome Dynamics Reveals the System-wide Regulatory Networks

    Masaaki Oyama, Hiroko Kozuka-Hata, Shinya Tasaki, Kentaro Semba, Seisuke Hattori, Sumio Sugano, Jun-ichiro Inoue, Tadashi Yamamoto

    MOLECULAR & CELLULAR PROTEOMICS   8 ( 2 ) 226 - 231  2009.02  [Refereed]

     View Summary

    Signal transduction systems are known to widely regulate complex biological events such as cell proliferation and differentiation. Because phosphotyrosine-dependent networks play a key role in transmitting signals, a comprehensive and fine description of their dynamic behavior can lead us to systematically analyze the regulatory mechanisms that result in each biological effect. Here we established a mass spectrometry-based framework for analyzing tyrosine phosphoproteome dynamics through temporal network perturbation. A highly time-resolved description of the epidermal growth factor-dependent signaling pathways in human A431 cells revealed a global view of their multiphase network activation, comprising a spike signal transmission within 1 min of ligand stimulation followed by the prolonged activation of multiple Src-related molecules. Temporal perturbation of Src family kinases with the corresponding inhibitor PP2 in the prolonged activation phase led to the down-regulation of the molecules related to cell adhesion and receptor degradation, whereas the canonical cascades as well as the epidermal growth factor receptor relatively maintained their activities. Our methodology provides a system-wide view of the regulatory network clusters involved in signal transduction that is essential to refine the literature-based network structures for a systems biology analysis. Molecular & Cellular Proteomics 8:226-231, 2009.

    DOI

  • Diversity of transplantation start sites may define increased complexity of the human short ORFeome

    Masaaki Oyama, Hiroko Kozuka-Hata, Yutaka Suzuki, Kentaro Semba, Tadashi Yamamoto, Sumio Sugano

    Molecular and Cellular Proteomics   6 ( 6 ) 1000 - 1006  2007.06

     View Summary

    Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5′-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5′- end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

    DOI PubMed

  • NF-kappaB activation in development and progression of cancer.

    Jun-Ichiro Inoue, Jin Gohda, Taishin Akiyama, Kentaro Semba

    Cancer science   98 ( 3 ) 268 - 74  2007.03  [International journal]

     View Summary

    Nuclear factor-kappaBeta (NF-kappaB) binds specifically to NF-kappaB-binding sites (kappaB sites, 5'-GGGRNNYYCC-3'; R, purine; Y, pyrimidine; N, any nucleotide) present in enhancer regions of various genes. Binding of various cytokines, growth factors and pathogen-associated molecular patterns to specific receptors activates NF-kappaB and expression of genes that play critical roles in inflammation, innate and acquired immunity, bone remodeling and generation of skin appendices. Activation of NF-kappaB is also involved in cancer development and progression. NF-kappaB is activated in cells that become malignant tumors and in cells that are recruited to and constitute the tumor microenvironment. In the latter scenario, the TLR-TRAF6-NF-kB pathways seem to play major roles, and NF-kappaB activation results in production of cytokines, which in turn induce NF-kappaB activation in premalignant cells, leading to expression of genes involved abnormal growth and malignancy. Furthermore, NF-kappaB activation is involved in bone metastasis. Osteoclasts, whose generation requires the RANK-TRAF6-NF-kappaB pathways, release various growth factors stored in bone, which results in creation of microenvironment suitable for proliferation and colonization of cancer cells. Therefore, NF-kappaB and molecules involved its activation, such as TRAF6, are attractive targets for therapeutic strategies against cancer.

    PubMed

  • AYUMS: An algorithm for completely automatic quantitation based on LC-MS/ MS proteome data and its application to the analysis of signal transduction

    Ayumu Saito, Masao Nagasaki, Masaaki Oyama, Hiroko Kozuka-Hata, Kentaro Semba, Sumio Sugano, Tadashi Yamamoto, Satoru Miyano

    BMC Bioinformatics   8  2007

     View Summary

    Background: Comprehensive description of the behavior of cellular components in a quantitative manner is essential for systematic understanding of biological events. Recent LC-MS/MS (tandem mass spectrometry coupled with liquid chromatography) technology, in combination with the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) method, has enabled us to make relative quantitation at the proteome level. The recent report by Blagoev et al. (Nat. Biotechnol., 22, 1139-1145, 2004) indicated that this method was also applicable for the time-course analysis of cellular signaling events. Relative quatitation can easily be performed by calculating the ratio of peak intensities corresponding to differentially labeled peptides in the MS spectrum. As currently available software requires some GUI applications and is time-consuming, it is not suitable for processing large-scale proteome data. Results: To resolve this difficulty, we developed an algorithm that automatically detects the peaks in each spectrum. Using this algorithm, we developed a software tool named AYUMS that automatically identifies the peaks corresponding to differentially labeled peptides, compares these peaks, calculates each of the peak ratios in mixed samples, and integrates them into one data sheet. This software has enabled us to dramatically save time for generation of the final report. Conclusion: AYUMS is a useful software tool for comprehensive quantitation of the proteome data generated by LC-MS/MS analysis. This software was developed using Java and runs on Linux, Windows, and Mac OS X. Please contact ayumsιms.u-tokyo.ac.jp if you are interested in the application. The project web page is http://www.csml.org/ayums/. © 2007 Saito et al; licensee BioMed Central Ltd.

    DOI PubMed

  • Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro.

    Tohru Takaki, Kazuhiro Fukasawa, Ikuko Suzuki-Takahashi, Kentaro Semba, Masatoshi Kitagawa, Yoichi Taya, Hiroshi Hirai

    Journal of biochemistry   137 ( 3 ) 381 - 6  2005.03  [International journal]

     View Summary

    D-type cyclin-dependent kinases (Cdk4 and Cdk6) regulate the G1 to S phase progression of the mammalian cell cycle. It has been suggested that Cdk4 and Cdk6 may have distinct functions in vivo, even though they are indistinguishable biochemically. Here we show that although these Cdks phosphorylate multiple residues in pRB, they do so with different residue selectivities in vitro; Thr821 and Thr826 are preferentially phosphorylated by Cdk6 and Cdk4, respectively. This raises the possibility different substrate specificities lead to their different roles in the regulation of cellular events. Furthermore, our results indicate the new concept that Cdk itself contributes to substrate recognition.

    PubMed

  • TIFAB inhibits TIFA, TRAF-interacting protein with a forkhead-associated domain.

    Takayuki Matsumura, Kentaro Semba, Sakura Azuma, Shuntaro Ikawa, Jin Gohda, Taishin Akiyama, Jun-ichiro Inoue

    Biochemical and biophysical research communications   317 ( 1 ) 230 - 4  2004.04  [International journal]

     View Summary

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals that lead to activation of NFkappaB and AP-1, which is essential for cell differentiation and establishment of the immune and inflammatory systems. TRAF-interacting protein with a forkhead-associated domain (TIFA) was identified as a TRAF6-binding protein that could link IRAK-1 to TRAF6 and then activate TRAF6 upon stimulation. We report identification of a TIFA-related protein, TIFAB, that inhibits TIFA-mediated activation of NFkappaB. TIFAB does not associate with members of the TRAF family but does bind TIFA. We analyzed the effect of TIFAB expression on the TRAF6/TIFA interaction by immunoprecipitation of TRAF6 and found that TIFA coprecipitated with TRAF6 was not changed. However, when we analyzed this interaction by immunoprecipitation of TIFA, we found that TIFAB significantly increased the amount of TRAF6 coprecipitated with TIFA. These findings suggest that TIFAB inhibits the TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA.

    PubMed

  • Cloning and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP-binding protein subfamily.

    Kosuke Ishikawa, Sakura Azuma, Shuntaro Ikawa, Yasuyuki Morishita, Jin Gohda, Taishin Akiyama, Kentaro Semba, Jun ichiro Inoue

    Gene   322   105 - 12  2003.12  [International journal]

     View Summary

    The developmentally regulated GTP-binding protein (DRG) subfamily is an uncharacterized member of the Obg family, an evolutional branch of GTPase superfamily proteins. GTPases act as molecular switches regulating diverse cellular processes. DRG2 and DRG1 comprise the DRG subfamily in eucaryotes. Although drg1 was first identified as a gene predominantly expressed during early development of the mouse central nervous system, comparative analysis of drg2 and drg1 expression during embryogenesis has never been reported, and the biochemical properties of the DRG family proteins remain to be elucidated. Thus, we first cloned Xenopus drg2 (Xdrg2) and examined the temporal and spatial expression patterns of Xdrg2 mRNA in comparison to those of Xdrg1. Both Xdrg2 and Xdrg1 are induced at late gastrula and subsequently increased during later stages of embryos (stage 13-41). Whole-mount in situ hybridization showed that Xdrg2 and Xdrg1 expression patterns are almost identical except that only Xdrg2 expression is detected in the stage 22 pronephric anlage. Strong transcripts of both genes are also observed at this stage in neural crest cells, blood islands, and developing eyes, and in brain, eyes, otic vesicle, branchial arches, pronephroses, spinal cord, notochord, head mesenchyme, and somites at stages 27 and 32. Northern blot analysis of adult tissues revealed that both genes are expressed highly in ovary and testis and rather moderately in other organs, except that Xdrg1 transcripts are scarcely detected in heart, lung, and liver. Accordingly, transcription or stability of Xdrg2 and Xdrg1 mRNAs may be regulated by different mechanisms. In addition, by generating recombinant XDRG2 and XDRG1 proteins, we found the RNA binding activity of these proteins in vitro. Our results suggest that the DRG proteins may play their physiological roles via RNA binding.

    PubMed

  • A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusion protein of desmoplastic small round-cell tumor.

    Emi Ito, Reiko Honma, Jun-ichi Imai, Sakura Azuma, Takayuki Kanno, Shigeo Mori, Osamu Yoshie, Jun Nishio, Hiroshi Iwasaki, Koichi Yoshida, Jin Gohda, Jun-Ichiro Inoue, Shinya Watanabe, Kentaro Semba

    The American journal of pathology   163 ( 6 ) 2165 - 72  2003.12  [International journal]

     View Summary

    Recurrent chromosomal translocations in neoplasms often generate hybrid genes that play critical roles in tumorigenesis. Desmoplastic small round-cell tumor (DSRCT) is an aggressive malignancy associated with the chromosomal translocation t(11;22)(p13;q12). This translocation generates a chimeric transcription factor, EWS-WT1, which consists of the transcriptional activation domain of the Ewing's sarcoma (EWS) protein and the DNA binding domain of the Wilms' tumor 1 (WT1) protein. One of the splice variants, EWS-WT1(-KTS) lacks three amino acid residues (Lys-Thr-Ser) in the DNA binding domain and transforms NIH3T3 cells. Therefore, it is likely that aberrant gene expression caused by EWS-WT1(-KTS) is involved in the malignant phenotype of DSRCT. Microarray analysis of 9600 human genes revealed that a gene encoding a tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1 (TALLA-1), was induced in EWS-WT1(-KTS)-expressing cell clones. This induction was EWS-WT1(-KTS)-specific, and more importantly, TALLA-1 protein was expressed in the three independent cases of DSRCT. Tetraspanin-family genes encode transmembrane proteins that regulate various cell processes such as cell adhesion, migration and metastasis. Our findings provide a novel insight into the malignant phenotype of DSRCT, suggesting that TALLA-1 is a useful marker for diagnosis and a potential target for the therapy of DSRCT.

    PubMed

  • Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling.

    Hiroshi Takatsuna, Hiroki Kato, Jin Gohda, Taishin Akiyama, Ayaka Moriya, Yoshinari Okamoto, Yuriko Yamagata, Masami Otsuka, Kazuo Umezawa, Kentaro Semba, Jun-Ichiro Inoue

    The Journal of biological chemistry   278 ( 14 ) 12144 - 50  2003.04  [International journal]

     View Summary

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.

    PubMed

  • Molecular cloning of a cyclin-like protein associated with cyclin-dependent kinase 3 (cdk 3) in vivo

    Masaaki Matsuoka, Yoshiharu Matsuura, Kentaro Semba, Ikuo Nishimoto

    Biochemical and Biophysical Research Communications   273 ( 2 ) 442 - 447  2000.07

     View Summary

    cdk3 has been considered to be rate-limiting for cell cycle progression of mammalian cells while its precise function remains to be elucidated, To assess cdk3 function, a cDNA coding for a cyclin-like protein (designated as ik3-1 from an interactor-1 with cdk3) was isolated with the yeast two-hybrid system using a cyclin-dependent kinase 3 (cdk3) cDNA as bait. p70(ik3-1) (a 70-kDa protein designated as p70(ik3-1)) seems to belong to the cyclin family as its C-terminal domain composed of 124 amino acids resembles the highly conserved cyclin box. Coimmunoprecipitation indicated that p70(ik3-1) binds to p35(cdk3) in vivo. The ik3-1 gene may belong to a multigene family and is highly conserved during evolution. mRNA expression of ik3-1 was low in the early G1 phase, upregulated during G1 progression, maximal at a mid-late G1 point, and declined gradually thereafter, suggesting that it may work mainly in G1 phase. (C) 2000 Academic Press.

    DOI PubMed

▼display all

Misc

  • Investigation of Cancer Reclassification by the HIF1 downstream genes.

    中道和也, 中山淳, 仙波憲太郎, 仙波憲太郎

    日本分子生物学会年会プログラム・要旨集(Web)   44th  2021

    J-GLOBAL

  • Analysis of microheterogeneity in the breast cancer orthotopic xenograft model

    中山淳, 中山淳, 仙波憲太郎, 仙波憲太郎, 山本雄介

    日本がん転移学会学術集会・総会プログラム抄録集   30th  2021

    J-GLOBAL

  • Establishment of novel brain metastasis evaluation system in HER2-positive breast cancer

    黒岩由佳, 黒岩由佳, 中山淳, 中山淳, 山本雄介, 仙波憲太郎, 仙波憲太郎

    日本がん転移学会学術集会・総会プログラム抄録集   30th  2021

    J-GLOBAL

  • 溶骨性luminal乳がん細胞株の性状解析

    韓宇軒, 韓宇軒, 中山淳, 中山淳, 二口充, 伊藤恵美, 渡辺慎哉, 仙波憲太郎, 仙波憲太郎

    日本癌学会学術総会抄録集(Web)   80th  2021

    J-GLOBAL

  • Signal analysis of lung metastatic breast cancer cell established by orthotopic xenograft

    林祐介, 林祐介, 中山淳, 中山淳, 山本瑞生, 井上純一郎, 山本雄介, 仙波憲太郎, 仙波憲太郎

    日本分子生物学会年会プログラム・要旨集(Web)   44th  2021

    J-GLOBAL

  • Analysis of brain metastasis in HER2-positive breast cancer cell lines by intracranial injection

    黒岩由佳, 中山淳, 仙波憲太郎, 仙波憲太郎

    日本がん転移学会学術集会・総会プログラム抄録集   29th  2020

    J-GLOBAL

  • Characterization of Luminal High-Osteolytic Breast Cancer Cell Lines

    韓宇軒, 中山淳, 林祐介, 二口充, 仙波憲太郎, 仙波憲太郎

    日本がん転移学会学術集会・総会プログラム抄録集   29th  2020

    J-GLOBAL

  • Analysis of growth mechanism and drug resistance in highly lung-metastatic breast cancer cell lines

    林祐介, 中山淳, 仙波憲太郎, 仙波憲太郎

    日本がん転移学会学術集会・総会プログラム抄録集   29th  2020

    J-GLOBAL

  • 脳組織中での増殖能に基づくHER2陽性乳がん細胞株の分類

    黒岩由佳, 黒岩由佳, 中山淳, 中山淳, 足立ちひろ, 井上貴文, 山本雄介, 渡辺慎哉, 仙波憲太郎, 仙波憲太郎

    日本分子生物学会年会プログラム・要旨集(Web)   43rd  2020

    J-GLOBAL

  • 尾動脈注射手法を用いた溶骨性luminal乳がん細胞株の樹立と性状解析

    韓宇軒, 韓宇軒, 中山淳, 中山淳, 林祐介, 鄭盛文, 二口充, 伊藤恵美, 渡辺慎哉, 仙波憲太郎, 仙波憲太郎

    日本分子生物学会年会プログラム・要旨集(Web)   43rd  2020

    J-GLOBAL

  • PHLDA3遺伝子とMEN1遺伝子による膵臓神経内分泌腫瘍抑制機構の解明(Unraveling the mechanisms of pancreatic neuroendocrine tumorigenesis using a new mouse model)

    陳 ヨ, 岩淵 禎弘, 清野 透, 間木 重行, 新井 康仁, 横山 明彦, 岡田 眞里子, 橋本 真一, 仙波 憲太郎, 大木 理恵子

    日本癌学会総会記事   78回   P - 1177  2019.09

  • Establishment and Characterization of Luminal Osteolytic Breast Cancer Cell Using Intra-Caudal Arterial Injection

    韓宇軒, 中山淳, 中山淳, 中山淳, 二口充, 伊藤恵美, 渡辺慎哉, 仙波憲太郎, 仙波憲太郎

    日本癌学会学術総会抄録集(Web)   78th  2019

    J-GLOBAL

  • HER2陽性乳癌治療薬開発標的としての新規Rac1活性化機構の解明

    前川大志, 前川大志, 村上朱里, 村上朱里, 川合克久, 中山淳, 荒木伸一, 仙波憲太郎, 田口友彦, 亀井義明, 高田泰次, 東山繁樹, 東山繁樹

    日本生化学会大会(Web)   92nd  2019

    J-GLOBAL

  • 多臓器転移誘導遺伝子HNF1Bの同定と機能解析

    中山淳, 二口充, 仙波憲太郎, 仙波憲太郎

    日本分子生物学会年会プログラム・要旨集(Web)   42nd  2019

    J-GLOBAL

  • Negative regulation of NF-kappa B signaling by Tobl in breast cancer

    Miho Tokumasu, Mizuki Yamamoto, Jyun Nakayama, Kentaro Semba, Jyunichiro Inoue, Tadashi Yamamoto

    CANCER SCIENCE   109   205 - 205  2018.01

    Research paper, summary (international conference)  

  • エンドソーム膜のホスファチジルセリンを介したYAPの活性化機構の解明

    松平 竜之, 向井 康治朗, 野口 大心, 長谷川 純矢, 八田 知久, 家村 俊一郎, 夏目 徹, 宮村 憲央, 仁科 博史, 中山 淳, 仙波 憲太郎, 冨田 拓哉, 村田 茂穂, 新井 洋由, 田口 友彦

    生命科学系学会合同年次大会   2017年度   [1P - 0321]  2017.12

  • Multi-organ metastasis誘導遺伝子HNF1Bの同定と機能解析

    中山 淳, 松井 貴香, 藤元 次郎, 二口 充, 伊藤 恵美, 渡辺 慎哉, 仙波 憲太郎

    日本癌学会総会記事   76回   J - 3104  2017.09

  • 尾動脈注射手法を用いたLumina1乳がん骨高転移株の作製

    鄭盛文, 中山淳, 中山淳, 二口充, 仙波憲太郎, 仙波憲太郎

    日本がん転移学会学術集会・総会プログラム抄録集   26th  2017

    J-GLOBAL

  • 膵臓がん細胞においてNuclear Factor-κB inducing kinase(NIK)はNF-κBの恒常的活性化と細胞増殖に寄与する(NIK is involved in constitutive activation of NF-κB and proliferation in pancreatic cancer cells)

    仁科 隆史, 山口 憲孝, 仙波 憲太郎, 井上 純一郎

    日本癌学会総会記事   68回   229 - 229  2009.08

    Research paper, summary (national, other academic conference)  

  • チロシンリン酸化プロテオームのシグナル動態解析によるSrcファミリー制御ネットワークの解明(Time-resolved description of tyrosine-phosphoproteome signaling dynamics reveals Src family-mediated global regulatory networks)

    秦 裕子, 尾山 大明, 田崎 真哉, 仙波 憲太郎, 服部 成介, 菅野 純夫, 井上 純一郎, 山本 雅

    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   81回・31回   4T25 - 5  2008.11

  • NF-kappa B activation in development and progression of cancer

    Jun-ichiro Inoue, Jin Gohda, Taishin Akiyama, Kentaro Semba

    CANCER SCIENCE   98 ( 3 ) 268 - 274  2007.03

    Book review, literature introduction, etc.  

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    Nuclear factor-kappa B (NF-kappa B) binds specifically to NF-kappa B-binding sites (kappa B sites, 5'-GGGRNNYYCC-3'; R, purine; Y, pyrimidine; N, any nucleotide) present in enhancer regions of various genes. Binding of various cytokines, growth factors and pathogen-associated molecular patterns to specific receptors activates NF-kappa B and expression of genes that play critical roles in inflammation, innate and acquired immunity, bone remodeling and generation of skin appendices. Activation of NF-kappa B is also involved in cancer development and progression. NF-kappa B is activated in cells that become malignant tumors and in cells that are recruited to and constitute the tumor microenvironment. In the latter scenario, the TLR-TRAF6-NF-kappa B pathways seem to play major roles, and NF-kappa B activation results in production of cytokines, which in turn induce NF-kappa B activation in premalignant cells, leading to expression of genes involved abnormal growth and malignancy. Furthermore, NF-kappa B activation is involved in bone metastasis. Osteoclasts, whose generation requires the RANK-TRAF6-NF-kappa B pathways, release various growth factors stored in bone, which results in creation of microenvironment suitable for proliferation and colonization of cancer cells. Therefore, NF-kappa B and molecules involved its activation, such as TRAF6, are attractive targets for therapeutic strategies against cancer.

    DOI

  • High-throughput temporal analysis of phosphotyrosine-dependent signaling networks by gel-free quantitative proteomics

    H. Kozuka-Hata, M. Oyama, S. Tasaki, K. Semba, S. Sugano, T. Yamamoto

    MOLECULAR & CELLULAR PROTEOMICS   5 ( 10 ) S83 - S83  2006.10

    Research paper, summary (international conference)  

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Awards

  • 日本癌学会奨励賞

    1988  

Research Projects

  • 正常―がん細胞間作用に基づくがん防御機構の化学生物学的解析

    日本学術振興会  科学研究費助成事業 挑戦的研究(萌芽)

    Project Year :

    2021.07
    -
    2024.03
     

    仙波 憲太郎

  • Screening for genes involved in efficient reprogramming directly into mammary stem cells

    Project Year :

    2015.04
    -
    2017.03
     

     View Summary

    We succeeded in immortalization of reporter mouse mammary epithelial cells (MECs), which allow us to screen genes involved in dedifferentiation, efficiently. 115 genes, which had been first under expectation as associated genes with dedifferentiation, unfortunately did not activate reporter cells. Thus, there was a need to evaluate larger gene sets. Then we developed a gene trap system for expression of genes covering more broad set including non-coding RNA using a transposon system. This is powerful technique one because there is no need to prepare cDNA libraries. Using this convenient and efficient system, we isolated 4 positive clones. Among them, we identified a candidate gene possibly involving regulation of stemness, and now are analyzing its function both in vitro and in vivo. Moreover, by applying the technique of the trap method, various technologies for evaluating the function of genes regulating stemness were also developed

  • Generation of mammary stem cells by direct reprogramming

    Project Year :

    2013.04
    -
    2015.03
     

     View Summary

    We constructed gene expression vector systems, which allow transient expression of slug/sox9 genes known to force mammary epithelial cells to dedifferentiate into mammary stem cells. We observed, as reported, that slug/sox9 expressed MEC reconstituted proper mammary ducts when transplanted, although the efficiency was quite low. To solve this problem, we changed donor mice to those expressing rtTA, established culture technology for maintaining mammary stem cells (MaSC), and screened genes enhancing efficiency of dedifferentiation. Among them, we successfully established the MaSC culture technology. In addition, we newly produced a reporter mouse for stemness. With these technologies, we now established screening system of genes enhancing dedifferentiation, easily, rapidly, and efficiently. However, we have not yet discovered any new direct reprogramming genes strongly leading to produce MaSCs

  • Development of experimental techniques for the analysis of amplicomics

    Project Year :

    2011.04
    -
    2014.03
     

     View Summary

    Cancer is a disease caused by mutations in genes. We carry two individual genes transmitted from father and mother, but in cancer cells, number of some genes is aberrantly increased by so-called "gene amplification". Accumulating evidence indicates that those genes, "oncogenes" can be used as target genes for diagnosis and therapy. Our research group has been focusing on breast cancer in which gene amplification is frequently observed and we have succeeded in developing several experimental procedures to discover oncogenes. For example, we established a protocol which enable us to efficiently introduce genes into normal cells to test transforming activity, namley whether or not they generate cancer cells. Finally, we found some genes which may serve as molecular diagnosis and targeted therapy for cancer. Furthermore, we performed functional analysis to know "why they make cancer"

  • srcファミリー遺伝子fynのリンホーマ並びに正常リンパ球における発現と機能

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    srcファミリー遺伝子fynの発現制御機構と、その産物p59^<fyn>チロシンキナーゼの生理的機能を解析し、以下の知見を得た。(1)発現:1prマウスで腫大リンパ節を構成する異常T細胞においては、p59^<fyn>キナーゼの活性が10倍亢進しており、これはfyn mRNAの過剰発現による。正常T細胞におけるfyn mRNAの誘導発現は、細胞内Ca^<++>レベル(up)とcAMPレベル(down)により調節を受ける。この誘導発現がtransientであるのに対し、1prT細胞の発現はconstitutiveであり、発現を継続維持させる内在性因子が作働しているものと思われる。今後、更に両者のメカニズムを解析してfyn遺伝子の発現に関与する調節因子(群)を同定したい。Tリンパ球以外にもB前駆細胞や、TPAにより分化したマクロファージにおいてもfynの強い発現を認めた。こうしたstageー及びlineageーspecificityを有するfyn遺伝子発現の生通的意義の解明も次年度以降の課題である。(2)機能:活性化T細胞においてp59^<fyn>がCーキナーゼによって燐酸化されることが判明し、p59^<fyn>キナーゼも、いわゆるキナーゼサーキットの一員として活性化シグナルの伝達に機能している可能性が示された。p56^<lck>とのアナロジーとして、p59^<fyn>が細胞内で他の分子種(細胞表面レセプター及び基質分子)と機能的複合体を形成しているものと考えられるが、そうした会合分子の候補として、分子量75kdの蛋白分子が見出された。現在、この分子を同定しつつある。今年度、活性型及び不活性型fynDNA更にはアンチセンスオリゴヌクレオチドを作成した。これらを種々の機能を有する免疫系細胞に導入してその機能変化を解析することによって、p59^<fyn>キナーゼのシグナル伝達系における役割を推定し得るものと期待している。新たに作成した活性型fyn遺伝子を導入したトランスジェニックマウスの解析結果からも有意義な知見が得られるはずである

  • がん遺伝子の検出と機能解析

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    本年度、特に進展のみられた成果は次の3点である。1.srcファミリ-に属する細胞質チロシンキナ-ゼの作用について研究をすすめ、それらの多くが血球系細胞で発現していることから、本年度はfynとT細胞、lynとB細胞、fgrと骨ずい系細胞の関係を解析した。fyn蛋白質は遺伝的にリンパ節腫張がおこるlpr/lpr或いは、gld/gldマウスのCD^-_4CD^-_8T細胞で過剰発現を示していることを見出した。一方、正常マウスのT細胞においては、増殖刺戦によってfynが上昇する。両者の作用の異同についてはこれからの問題である。lyn蛋白質がB細胞特異的に発現していることは昨年報告したが、lynはIgM発現B細胞株であるWEHI231を緩やかな条件で可溶化した所、sIgMと免疫できることがわかった。lynは細胞に抗IgM抗体を作用させるとdown-regulationがかかることから、lynとIgMが相互作用していることが推定された。fgr蛋白質は末梢血由来のマクロファ-ジ、顆粒球、LGL等の細胞で発現している。HL-60やTHP-1細胞を分化誘導したときにfgrの発現がみられること、よく符号することと考えあわせ分化形質との関連性を検討する必要がある。2.転写調節:yesとlynを中心にその上流の転写調節を検討した。yesはその発現が比較的非特異的なことを裏付けるようにGCBOXが主体と考えられる一方、lynはB細胞に特異的なオイタマ-配列がみられるが詳細は今後の検討課題である。erbA関連のear-3遺伝子はGREに作用することとリンガンドが血清中にあることが明らかとなった。又ear-1はTRE(T3-responsible element)に作用することが分かった。3.earb-2の活性化機構及びヒトがんとの関係:eabB-2蛋白質の構造と機能の関係を解析し、キナ-ゼドメインのチロシン残基のリン酸化が活性上昇をもたらすこと、C末端の約200アミノ酸がチロシンキナ-ゼ活性抑制的に調節していること等を見出した

  • がん遺伝子の機能解析

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    リンパ球の抗原受容体と物理的に会合しているLynやFynが、抗原受容体を介するシグナル伝達反応に於て機能していることを示した。そして、Lynからのシグナル伝達系には、分子量約70,000のチロシンリン酸化された蛋白質やPIー3キナ-ゼが関わっていること、また、FynからのシグナルがSH2とCD3ζ鎖の相互作用により下流に伝えられることを示唆した。さらに、脳においてFynは胚ならびに生直後そして成体の全てで、海馬等の神経細胞で発現している事を示した。更に、免疫グロブリン遺伝子エンハンサ-支配下のlynならびにfyncDNAをtransgeneとして有するマウスを作成し、B細胞の機能・分化についての解析を進めている。さらに、gene targeting法により、fyn遺伝子を欠損するマウスを共同研究で得ているし、lyn遺伝子についても進行中である。Yes蛋白質については、アミノ末端近傍で細胞周期依存的にリン酸化されることを見いだし、その意義について検討中である。ErbBー2蛋白質については、子牛血清中に見いだした特異的なキナ-ゼ活性促進因子の精製を進め、アルファフェトプロテインに富む画分にその活性をが存在することを見いだした。さらに数種のステロイドホルモンがErbBー2蛋白質に直接結合して、そのキナ-ゼ活性を亢進させること、そしてErbBー2蛋白質の標的蛋白質としてPIー3キナ-ゼと蛋白質とキナ-ゼ活性を有する分子を予備的に見いだしている。遺伝子発現調節機構の解析に関しては、lyn遺伝子とcーerbBー2遺伝子について進展があった。まずp40^<tax>によりlyn遺伝子プロモ-タ-が活性化されることを明かにすると共にそのプロモ-タ-領域の詳細な解析を行なった。cーerbBー2プロモ-タ-に関しては、乳癌細胞株での転写活性上昇に関わる配列に結合する転写因子の候補を得ている

  • Srcファミリ-遺伝子fynのリンホ-マ並びに正常リンパ球における発現と機能

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    srcファミリ-遺伝子fynの発現制御機構と、その産物p59^<fyn>チロシンキナ-ゼ(fyn)の生理的機能を解析して以下の知見を得た。(1)発現:lprマウスで腫大リンパ節を構成する異常Tリンパ球においては、正常Tリンパ球に比べて約15倍のfyn mRNAの増大が認められるが、nuclear run on assayとactinomycinDを用いたfyn mRNA代謝速度の検定結果から、Iprリンパ球ではfyn nRNAのstabilityが増大していることが判明した。一方、活性化正常Tリンパ球に認められる一過性誘導発現では、核における転写量が増大していた。更に現在、前年度見だした前駆Bリンパ球におけるfynの高レベル発現がいずれの機序によるか検討している。(2)機能:免疫沈降法とin vitro kinase assayとを組み合わせることによって、FynがTリンパ球抗原レセプタ-(TcR)と会合していることを見出した。このことはTcRと共にdown modulationされることからも示唆され、またFynの5乃至10%がTcRに会合していた。TcRへの会合量はlprTリンパ球では正常の2.5倍であり、リンパ腺腫脹の形成との関連が示唆された。このlprTリンパ球においては、活性化Tリンパ球と同様にTcRのシグナル伝達分子CD3ζ鎖が構成的にチロシン燐酸化されているが、CD3ζ遺伝子とfynとのcoーtransfectionの系を用いて、CD3ζがFynの基質になり得ることが示された。更にlprTリンパ球でGAP(GTPaseーactivating protein)のチロシン燐酸化も判明し、Fynを介した伝達系の一端が明らかとなった。TcRに会合するFynの機能を知るため、現在更に、作成した種々の活性型及び不活性型fynを導入発現させた抗原特異的T細胞ハイブリド-マの反応性を解析している

  • がんの増殖と分化に関連する遺伝子の発現制御

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    私達は、がん細胞においてその発現制御様式が特異的に変化する遺伝子、がん細胞の増殖,分化を促進する遺伝子などに関して、その発現機構,作用機構の解析を行っている。サイトカインGーCSF,ILー6,ILー2は、本来の標的細胞に作用するばかりでなく、ある種のがん細胞(白血病細胞)などにも作用する。私達はGーCSF,ILー2受容体cDNAを単離し、これらの受容体が他のサイトカイン(ILー3,ILー4,ILー5やGMーCSF)受容体とともに、新しい受容体superfamilyを形成していることを示した。これらの受容体群の細胞外領域には、約200アミノ酸残基から成るお互いに相似した部分が存在するが、細胞内領域は似ていず、キナ-ゼなどの酵素活性領域も存在しないことから、全く未知の機構で増殖,分化のシグナルが細胞へ伝達されると考えられる。特に、T細胞のがん化などによりその発現が増加することが判明した<lyn>___ーなどの<src>___ー family遺伝子との相互作用が注目される。一方、低分化型胃がんにおいて特異的に増幅している遺伝子Kー<sam>___ー,それと関連し、未分化胚細胞で発現しているNー<sam>___ー遺伝子の構造を解析したところ、これらは繊維芽細胞増殖因子(FGF)受容体と類似しており、ヘパリン結合性増殖因子に対する受容体群を形成していることが示された。ある種のがん細胞は、本来、誘導的に発現されるGーCSF,ILー6などの遺伝子を構成的に発現する。私達は、GーCSF,ILー6,Pー450,cー<lyn>___ー,ミオシンlight chain,MHC,cー<myc>___ー遺伝子の発現調節に関与しているcisーelementsを同定するとともに、GーCSF,ILー6,Pー450,ミオシンlight chainなどにおいては、これらcisーelementに結合する転写因子のcDNAを単離した。これらは、ロイシン・ジッパ-構造をもちC/EBPー1群に属する遺伝子、Zn^<2+>フィンガ-構造をもちSpl群に属するものなどに分別され、単量体、あるいは二量体として作用すると考えられる

  • がん遺伝子の機能解析

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    細胞性がん遺伝子(がん原遺伝子)産物の中でも、特にチロシンキナ-ゼ活性を有する蛋白質の機能解析と、それら遺伝子の発現制御機構に関する研究を行なった。(1)チロシンキナ-ゼファミリ-蛋白質の機能解析。Lyn,Fyn,FgrとErbBー2の解析を進めた。Lynについては、Bリンパ球の抗原受容体であるsIgMならびにsIgDと会合していることを明らかにした。更に、LynがsIgM/D複合体を形成している30ー40kdと強く相互作用してそれらをin vitroで燐酸化することを示し、sIgを介するシグナル伝達に関与している可能性を指摘した。一方FynについてはTリンパ球抗原受容体と会合していることを明らかにすると共に、活性型FynをTリンパ球で発現させることにより、FynがT細胞活性化反応に関与しているという知見を予備的に得ている。Fgrについては、細胞障害活性を有する血球系細胞で発現していることを、蛋白質レベルで明らかにした。ErbBー2ついては、そのチロシンキナ-ゼ活性を促進する因子を血清中に見いだし、有力なリガンド候補と考えて精製を進めている。一方、活性型ErbBー2による細胞がん化反応が、キナ-ゼ活性欠損型のErbBー2によって抑制されることを示し、ErbBー2によるシグナル伝達反応にErbBー2蛋白質間の相互作用が重要であることを明らかにした。(2)転写調節機構の解析cーyes、lynプロモ-タ-の構造と機能について解析を進め、cーyesプロモ-タ-ではSpー1結合部位が転写活性に重要であることを示し、またlynについてはT細胞での発現抑制に関与する領域を見いだした。一方cーerbBー2遺伝子が乳がんなどで過剰発現する機構について、転写レベルでの予備的研究を開始している

  • Tリンパ球抗原レセプターに会合するチロシン燐酸化酵素Fynの機能

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    自己免疫マウスMRL/lprで異常増生しているTリンパ球において、チロシンキナーゼFynの発現が亢進し、抗原レセプターとの会合量が3倍に増加していることを報告したが、本年度は、こうしたTリンパ球における種々なシグナル伝達分子群のチロシン燐酸化を調べた。その結果、FynとGAP(GTPase activating protein)が恒常的にチロシン燐酸化されていることが判明した。更に、免疫沈降法とキナーゼアッセイとを組合わせることよって、FynとGAPとが会合していることが明らかになった。Fynが疾患の発症に関連することが、fyn遺伝子を消失させた(gene targetting)MRL/lprマウスの解析から示された。現在、自己免疫疾患及びリンパ腫の発症におけるFynの機能について解討している。前年度、抗原特異的T細胞ハイブリドーマに、正常型、活性型、不活性型及びSH2欠失型fyn遺伝子を導入して発現させると、正常型及び活性型Fyn変異体で抗原応答性が増大することを示した。本年度、Fynの機能解析を目的として、Fyn変異体における抗原刺激後の蛋白質分子群のチロシン燐酸化を調べた。その結果、PLCγ、p95^<vaw>及びMAPキナーゼのチロシン燐酸化が正常型及び活性型Fyn変異体で増大しており、その程度がほぼIL-2産生量と一致していた。以上の事実から、T細胞抗原受容体に会合するFynは、GAP、PLCγ、p95^<vaw>及びMAPキナーゼをチロシン燐酸化してその機能を修飾することによってTリンパ球の抗原応答性や分化に関わっているものと思われる。更に、本年度、クラスIIMHC遺伝子をマクロファージ上に強制発現させたトランスジェニックマウスを作成することで、Tリンパ球にアネルギー性寛容を導入し得たので、Fynと寛容との関連を解析することが可能になった

  • 細胞癌化における蛋白質リン酸化

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    1.estrogen(E2)がErbBー2のチロシンキナ-ゼ(PTK)活性を活性化しdown regulationをひきおこすことを見出した。2.B細胞抗原受容体を刺激することによりLynが活性化されること、Fyn過剰発現によりT細胞受容体を介した情報伝達反応が促進されることが示された。3.cーSrcのTyrー527をリン酸化化するPTK(CSK)のcDNAクロ-ニングに成功した。4.Crk蛋白質がリン酸化チロシンを有する蛋白質と特異的に結合することを示した。5.ラット肝からのAJの単離精製に成功し、Src,Yes,Lyn蛋白質が局在していることを見出し、さらにYes蛋白質と複合体を形成する分子の候補を同定した。またAJのリン酸化チロシンが細胞周期により変動するという結果を得た。6.MAPキナ-ゼの完全精製と、cDNAクロ-ニングに成功した。またMAPキナ-ゼがG0/G1期だけでなくS期およびM期にも活性化し細胞質のみでなく核にも局在する事を明らかにした。Rafー1キナ-ゼについては、活性化型は正常化型と比べ膜や核への局在が多いこと、増殖刺激や形質転換によってリン酸化が増大することを見出した。新しい遺伝子検索細胞SHOKを用いて単離したcot遺伝子が新しいタイプのSーG2期に活性化するPS/TKをコ-ドしていることを明らかにした。またヒトcーcot遺伝子を単離、構造決定し産物を同定した。7.Jun結合蛋白質のリン酸化状態が形質転換に伴い、顕著に変化することを見い出した。cdk2キナ-ゼおよびMAPキナ-ゼがRBキナ-ゼである可能性を示唆する実験結果を得た。またRBと結合する細胞蛋白質をコ-ドするcDNAのクロ-ニングに成功した。8.高品質cDNAライブラリ-の作製、大腸菌、酵母への効率の良い遺伝子導入法を開発し酵母の細胞周期変異を相補するヒト遺伝子(weel,cdc25)を単離した

  • Information Exchange on Cancer Research with North American

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    Recent progress on cancer research has revealed that cancer is induced and developed by subverted expression and/or damage of multiple genes. Especially genes called oncogenes and antioncogenes are deeply involved in cancer. These genes, encoding signal transduction molecules for cell growth and regulators of cell cycle, are activated by point mutations and amplifications or missing by deletion in cancer cells. The other genes that are involved in cell-to-cell interaction and cell-to-substrate interaction are likely to be important in metastasis.The knowledge on a molecular basis on cancer is going to be applied for diagnostic and therapeutic benefits. Data on three dimensional structures of the signaling proteins or oncogene products that are activated in cancer are useful to design a molecule that would inhibit growth of cancer cells. Genes that are missing or not properly functioning would be amended by gene therapy. These challenging methodologies would be promising when applied in cancer therapy in combination with well established protocols.Basic cancer research and application research that include human genome project, gene manupilation in experimental animals, and development of gene therapy have been highly progressed in the countries in north America, Canada and United States. Progress in Japanese cancer research is also remarkable in the last decade, allowing effective collaboration, information exchange between the scientists of this field in north America and Japan. Therefore, in this International Scientific Research Program, we sent 51 Japanese scientists during the last three years to promote information exchange and collaboration and for presentation of their progresses in the international meetings in north America. Representative topics for information exchange were following.Oncogenes/protooncogenes Antioncogenes TranscriptionSignal transduction Cell cycle regulation Viral oncologyCell adhesion and metastasis Apoptosis Genetic instabilityChemical carcinogenesis Gene manupilation in animals Structural biologyMolecular diagnosis Gene therapy Immuno-therapyRadiation therapy Molecular etiolog

  • 小児腎腫瘍の発症メカニズムの解析

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    ウィルムス腫瘍は小児腎腫瘍のうち約90%を占める代表的な悪性腫瘍である。本研究はこの腫瘍の発症に関わる遺伝子として単離されたWT1遺伝子を分子生物学的手法で解析することにより、腫瘍の発症機構を解明することを目指している。本年度は以下のことを明かにした。1.WT1蛋白質はAキナーゼによりDNA結合ドメイン内のSer-365,Ser-393がリン酸化されること、このリン酸化がWT1蛋白質のDNA結合活性および転写制御能を阻害することを証明した。即ち、ホルモン刺激などによって作動するcAMP-A-kinaseの経路によりWT1蛋白質の機能が抑制される可能性を見い出した。2.WT1蛋白質と結合する蛋白質のcDNAとして生殖器の発生に関与するSRYと相同性を示す新規SRY関連遺伝子を単離した。WT1遺伝子は生殖器の発生異常を伴うDenys-Drash症候群の患者において高率に変異が見い出される。我々はWT1蛋白質がこのような遺伝子産物との結合を介して生殖器の発生を制御している可能性を考えてこの新規遺伝子の解析を進めている。3.アフリカツメガエルからWT1遺伝子をクローニングしてその構造および発現を解析した。その結果、WT1遺伝子は脊椎動物にわたって高度に保存されており、前腎と呼ばれる最も原始的な腎臓が発生する初期から発現することが明らかになった。カエル卵のアニマルキャップに腎管を誘導する系を用いてWT1遺伝子の発現を検討したところ、腎管を誘導した場合にのみWT1遺伝子の発現が認められた。以上のことから、WT1遺伝子は脊椎動物にわたって腎臓の発生に関与する可能性が示唆された。現在、この系がWT1蛋白質の機能アッセイ系に利用できるかどうかを検討する目的でdominant negative型WT1遺伝子をカエル卵に発現させて前腎の発生に与える影響を調べている

  • Molecular mechanism of sex determination and its disease

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    In humans, as in other mammals, sex determination is controlled by a dominant switch termed TDF for Testis Determining Factor. The SRY gene is thought to be the TDF, which encodes a transcription factor with one HMG box as a DNA binding domain. Mutations in the SRY HMG box have been identified in 15% cases of XY sex reversal in humans. Introduction of mouse SRY gene (Sry) into XX female mice induced testis differentiation and subsequent male development. However, little is known about mechanism of transcriptional regulation by SRY. WT1 mutations have frequently been observed in Denys-Drash syndrome (DDS) patients with urogenital malformation.During analysis of WT1-associated proteins, we found that WT1 bound to Sox30, which encodes a novel transcription factor with one HMG box. Further analysis showed that WT1 bound to its HMG box. This observation prompted us to analyze interaction between WT1 and SRY, both of which are expressed in the somatic cells of early gonads. We showed the following results:i) WT1 binds to SRY in vitro and in cultured cells.ii) WT1 and SRY synergistically activate transcription from a promoter that contains SRY binding sequence and also from the SRY promoter.iii) WT1 mutants found in DDS, however, did not show this activity.iv) WT1 is recruited on a SRY-binding sequence in a SRY-dependent manner, while recruitment of DDS mutants is significantly reduced.These observations suggest that WT1 and SRY interaction plays an important role in early gonadal development and its disease. Recently we have established cell lines which express both WT1 and SRY. We are currently analyzing the expression profile of this cell line, which will reveal target genes regulated by WT1 and SRY

  • 転写制御の異常と細胞の増殖・分化

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    転写制御の異常は細胞周期制御の破綻を導き、時には、癌化を誘導すると考えられているが、不明な点が多い。本研究では、「転写因子の活性制御機構及び、転写因子の異常によって生じる細胞機能の破綻を分子レベルで解明する」ことを目指す。特にNFκB活性化のがん化における役割、TRAF6によるNFκB活性化シグナル伝達機構及びTRAF6シグナルによる細胞増殖・分化の誘導機構の解明を目的として今年度は以下の結果を得た。1)HTLV-1 Taxタンパク質によるNFκB活性化にK63型ユビキチン付加酵素Ubc13およびTAK1活性化は必要ない。従ってTaxはIL-1やLPS等のシグナルとは異なる機構でIKKを活性化する。また、HTLV-1感染細胞ではTax依存的NFκB活性化とTax非依存的かっ恒常的NFκB活性化の両者が起こっている。2)TRAF6による細胞増殖機構に関与する考えられるDRG1とDRG2を同定した。さらにDRG1とDRG2の発現量をユビキチン化反応の阻害により維持する特異的結合タンパク質DFRP1とDFRP2を同定した。3)TRAF6がTLR3以外のTLRの下流で必須な役割を果たしていることを明らかにした。またTRAF6欠損により胸腺上皮細胞の分化不全が起こりその結果自己抗体が産生されることを示した。即ちTRAF6によるNFκBの活性化が自然免疫と獲得免疫において重要な役割を果たし、がん免疫に必須であることが明らかとなった

  • Molecular mechanism of sex determination and its disease

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    In humans, as in other mammals, sex determination is controlled by a dominant switch termed TDF for Testis Determining Factor. The SRY gene is thought to be the TDF, which encodes a transcription factor with one HMG box as a DNA binding domain. Mutations in the SRY HMG box have been identified in 15% cases of XY sex reversal in humans. Introduction of mouse SRY gene (Sry) into XX female mice induced testis differentiation and subsequent male development. However, little is known about mechanism of transcriptional regulation by SRY. Mutations of another transcription factor, WT1, are frequently observed in Denys-Drash syndrome (DDS) patients with urogenital malformation.During analysis of WT1-associated proteins, we found that WT1 bound to Sox30, which encodes a novel transcription factor with one HMG box. Further analysis showed that WT1 bound to its HMG box. This observation prompted us to analyze interaction between WT1 and SRY, both of which are expressed in the somatic cells of.early gonads. We published a paper describing the following results in Oncogene. i) WT1 binds to SRY in vitro and in cultured cells and this interaction is mediated by the zinc finger domain of WT1 and the HMG box of SRY. ii) WT1 and SRY synergistically activate transcription from a promoter that contains SRY binding sequence and also from the SRY promoter. WT1 mutants found in DDS, however, did not show this activity. iii) WT1 is recruited on a SRY-binding sequence in a SRY dependent manner, while recruitment of DDS mutants is significantly inhibited.These observations suggest that WT1 and SRY interaction plays an important role in early gonadal development and its disease

  • 白血病細胞における細胞骨格・転写・細胞周期の制御異常

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    t(8;21)転座、inv(16)逆位はそれぞれ、Runx1-MTG8及びPEBP2beta-SMMHCキメラ遺伝子を生成する。AMLとしては各々、M2及びM4サブタイプの形態学的表現型を示す。上記の2つ、及びその他の染色体異常を伴うAML、合計54症例を用いて遺伝子発現プロファイリングを実施した。コントロールを適切に取ることにより、lineage/stageに依存せずに発現変動を示すと考えられる遺伝子群を抽出した。t(8;21)とinv(16)に共通して発現上昇する遺伝子群も存在したが、t(8;21)特異的に又はinv(16)特異的に発現上昇する遺伝子群も検出することができた。2つのキメラ蛋白は、正常のRunx1/PEBP2beta転写因子機能に対してドミナント・ネガティブに作用することが知られているが、それのみによって2つの型の白血病細胞における遺伝子発現パタンを説明できるものではないことが示唆された。WT1の4種類のアイソフォームおよびDSRCT (desmoplastic small round cell tumor)の発症に関わるとされるEWS-WT1キメラがん遺伝子の発現細胞からRNAを調製し、20,000遺伝子を網羅するマイクロアレイを用いて発現プロファイルを作成した。これにより、WT1のアイソフォームとEWS-WT1の各々に特異的に誘導される遺伝子群を同定した。EWS-WT1発現細胞では、血管新生抑制因子BAI-1と接着と運動にかかわるTALLA-1に着目して解析を進めた。Jurkat細胞において、抗TALLA1抗体は細胞どうしの接着性を著しく促進させること、抗CD3抗体と協調的にERKのリン酸化を促進することを示した

  • Identification and functional analysis of novel oncogenes involved in the breast cancer genesis and promotion

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    The forkhead transcription factor FoxA1 is thought to be involved in mammary tumorigenesis. However, the precise role of FoxA1 in breast cancer development is controversial. We examined expression of FoxA1 in 35 human breast cancer cell lines and compared it with that of ErbB2, a marker of poor prognosis in breast cancer. We found that FoxA1 is expressed at high levels in all ErbB2-positive cell lines and a subset of ErbB2-negative cell lines. Down-regulation of FoxA1 by RNA interference significantly suppressed proliferation of ErbB2-negative and FoxA1-positive breast cancer cell lines. Down-regulation of FoxA1 also enhanced the toxic effect of Herceptin on ErbB2-positive cell lines through induction of apoptosis. Taken together, our present results suggest that FoxA1 plays an important role as a lineage-specific oncogene in proliferation of cancer cells derived from mammary luminal cells.Almost 20% of cancer cells show constitutive NF-кB activity, which induce expression of several genes involved in tumor growth, metastasis and angiogenesis, however, the mechanism of the constitutive activation is unclear. To answer this question, we first evaluated basal NF-кB activities of 125 human cancer cell lines from various tissues with electorophoretic mobility shift assays. We are analyzing the mechanism using the cell lines showing high NF-кB activity

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Specific Research

  • HER2陽性乳がん細胞株における骨転移・肺転移能の評価

    2021  

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    本研究の目的は、尾動脈移植手法(骨転移)および尾静脈移植手法(肺転移)を用いて所有する9種類のHER2陽性乳がん細胞株の骨転移能および肺転移能を評価し、転移モデルとしての有用性と各細胞株の転移特性を明らかにすることである。成果として、UACC893, MDA-MB-453, HCC202細胞は骨転移を起こすこと、肺転移効率は総じて低いことを明らかにした。遺伝子発現解析より、新たな予後マーカーを見出した。以上の結果をClinical &amp; Experimental Metastasis (https://doi.org/10.1007/s10585-022-10150-1)に発表した。

  • 受容体型チロシンキナーゼの切断による抗体医薬耐性獲得機構の解明とその克服への試み

    2020  

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    我々は膜貫通型セリンプロテアーゼ(TTSP)であるTMPRSS4がERBB2を切断・活性化させることで腫瘍形成に寄与することを明らかにした。プロテアーゼ阻害剤はコロニー形成を抑制したが、現在臨床に使用されているプロテアーゼ阻害剤では、in vivoの腫瘍抑制効果がない。そこで、TMPRSS4に特異的で強く活性を抑制する化合物の探索を目的としたスクリーニング系を検討した。ERBB2のN末端に容易に定量可能なタグタンパク質を融合し基質として用いることで、TMPRSS4に切断され遊離した細胞外ドメインが充分な感度で検出され、これを用いた高感度のスクリーニング系を構築することができた。

  • 頭蓋内移植手法によるHER2陽性乳がんの脳転移機構の解明

    2020  

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    乳がんの中でも、HER2陽性乳がんは脳への転移を起こしやすい。本研究では、HER2陽性乳がんの脳組織中での増殖メカニズムを解明するため、9種類のHER2陽性乳がん細胞株の脳組織中での増殖能評価を行なった。その結果、脳での増殖が速い細胞株と遅い細胞株が存在することが明らかとなった。また、遺伝子発現解析により、脳での増殖が速い細胞株に共通の遺伝子発現パターンを検出した。その中にはHER2陽性乳がん患者の予後不良と相関する遺伝子が含まれており、これらの遺伝子はHER2陽性乳がんの予後マーカーとして応用できる可能性が示された。本研究成果は、HER2陽性乳がん脳転移のさらなる研究に有用であると考えられる。

  • 正常-がん細胞間作用によるがん細胞増殖抑制機構の化学生物学的解析

    2019  

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    本研究では、正常細胞―がん細胞間作用を作用標的として、変異KRAS遺伝子を発現する細胞(がん細胞のモデル)の増殖抑制を誘導する抗がん化合物の作用機序の解明を試みた。ヒット化合物について約30個の類縁体を検討した結果、構造活性相関が見出された。また、ヒット化合物を磁気ビーズに共有結合させた化合物ビーズを用いて、化合物特異的に結合したタンパク質のアフィニティー精製を行い、これらタンパク質をLC-MS/MSにより同定した。その結果、ヒット化合物に特異的に結合を示すタンパク質を複数同定した。今後、これらタンパク質をコードする遺伝子のノックダウン実験により、化合物との結合による機能変化と、正常細胞によるがん細胞の増殖抑制機構解明との関連の解明を進める。

  • 受容体型チロシンキナーゼの切断による抗体医薬耐性獲得機構の解明とその克服の試み

    2019  

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    新規ERBB2切断酵素として同定したプロテアーゼTMPRSS4について、ERRB2と協調した腫瘍悪性化及びERBB2を標的とする抗体医薬Trastuzumab に対する耐性への寄与について検討した。乳がんにおけるTMPRSS4の発現と予後の関係を解析した結果、HER2陽性サブタイプのみにおいてTMPRSS4の発現が予後の悪さと相関し、病態悪化への寄与が示唆された。また、ERBB2とTMPRSS4を発現する細胞株をマウスに移植しTrastuzumab に対する感受性試験を行ったところ、ERBB2のみを発現する細胞に比べ感受性が低下していた。この結果は、受容体型チロシンキナーゼを標的とする抗体医薬に対する耐性獲得機構の一端を明らかにするものである。

  • 空間トランスクリプトミクス技術を用いた転移能獲得に至る腫瘍内不均一性の動態解明

    2017   中山 淳

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    腫瘍組織における微小不均一性は、遺伝的な不均一性と微小環境の不均一性の2つから成り立っている。これを明らかにするためには、組織を細分化する技術と細分化した組織に対して解析を行う技術が必要である。私達は自動かつ高速に微小組織切片を採取することが可能なシステム(Yoda T et al., Sci. Rep., 2017)を応用し、ヒト乳がん細胞株MDA-MB-231を免疫不全マウスに移植することで作製した乳がん原発腫瘍に対して多量サンプリングを行った。さらに微小組織からのRNA抽出とRNA-seq解析を組み合わせることで、任意の微小部位における遺伝子発現発現を解析する空間トランスクリプトミクス解析技術を確立した。

  • 乳腺幹細胞への高効率なダイレクトリプログラミング因子の同定

    2017  

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    レポーターマウスの乳腺上皮細胞(MEC)の不死化に成功しスクリーニングが効率化した。当初期待していた115個の遺伝子には活性がなく、より大きな遺伝子セットを評価する必要があった。そこでアノテーションされていないnon-coding RNAなどを含む遺伝子セットを大量に評価可能なトランスポゾンを用いた遺伝子発現・同定技術を考案した。開発したベクターをレポーター細胞に導入し、4つの陽性クローンを得ることに成功した。その内1クローンから候補遺伝子が同定され、さらに解析を進めているところである。また、トラップ法の技術を応用し、幹細胞化遺伝子の機能評価のための各種技術を構築した。

  • 乳腺構造の微小環境を利用した癌遺伝子の解析システム

    2013  

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     本研究は癌の悪性化への微小環境の影響を解析するため、①乳腺再構築系を用いて、人工的に微小環境を変化させて癌化を誘導し、解析するシステムの開発、および、②乳管内に直接的に細胞を導入してDCISを形成させ、その腫瘍環境からの浸潤・転移などの悪性化を評価するシステムの開発を最終目的としている。 ①について繊維芽細胞などを加工した間質系と遺伝子改変乳腺上皮細胞の相互作用を観察する予定であったが、再構築させる乳腺側の幹細胞への遺伝子導入が、今後予定する多量の遺伝子スクリーニングには耐えられない効率であり、このシステム開発における大きな問題となっていた。そこで本年度は乳腺幹細胞を濃縮し、効率よく導入する方法の確立を最優先課題として検討した。セルソーターの利用を視野に、乳腺細胞を脂肪細胞・繊維芽細胞・血球細胞などから分離する技術を向上させた上、MACSとの組み合わせで効率よく幹細胞画分をソート可能なプロトコールを確立させた。また、感染させるウイルスのpackagingベクターの比を変えることで導入効率を向上させることに成功した。 ②では、乳癌サブタイプが明確で種類が豊富なヒト乳癌細胞株での解析を目指し、生着の試験としてヒト乳癌細胞株であるMCF7を免疫不全マウスであるrag2KOに導入したが、生着が著しく悪かった。皮下移植の検討で、免疫不全度がより重度のNOD/SCIDマウスの方が生着率が良いことが判明し、今後のレシピエントマウスの使用における重要な知見を得た。また、マウス乳腺から繊維芽細胞を採取し炎症性サイトカインで刺激した際にヒト乳癌細胞の癌幹細胞性の維持に重要なJAG1発現が亢進することや、乳腺上皮細胞においても妊娠を模倣したホルモン投与によってJAG1発現が誘導される事を明らかとし(文献1)、今後の解析における炎症や妊娠による乳腺微小環境の変化の重要性が示唆された。 また、①②に共通する腫瘍化後の経時変化の観察においてIVISの利用が可能となったため、用いる一連の細胞株にLuciferase遺伝子を導入した。また、rag2KOマウスはB6黒毛による発光吸収の改善のため、FBVマウスバックグラウンドのアルビノ化を進めている。

  • ローコストスクリーニングによる癌代謝異常の解析

    2012  

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    ゲノムワイドの遺伝子スクリーニングは生命現象の新たな制御系を発見するために必須の技術である。最近はこの目的で次世代シークエンサーが注目を集めているが、膨大なデータ量の創出とその解析に高額の研究費を要する。震災後、科学研究費においても危機的状況にある本邦においては、既存の技術や研究資源を統合する創意工夫を凝らしたローコストスクリーニングシステムを構築することが必要である。具体的には、近年注目を集めている癌の代謝異常に注目し、申請者の持つ遺伝子発現プロファイルと種々の公開データベースを横断的に解析し、三次元細胞培養系を組み合わせることで、ローコストの新規細胞制御遺伝子の探索システムを構築し、癌における代謝異常に関わる遺伝子をスクリーニングする。申請者はこれまでに独自に作成した遺伝子発現データベースを用いて、乳癌の新たな遺伝子増幅領域(アンプリコン)を推定するし(Ito et al, FEBS Lett. 581,3909-14, 2007)、複数のアッセイ系でアンプリコンに存在する癌の増殖や浸潤に関わる遺伝子の同定に成功している。また、細胞分裂に応じて発現変動する公共の遺伝子データベース等を横断的に解析したin silicoスクリーニングとsiRNAによる機能解析により、細胞分裂を制御する新たな遺伝子を20種以上同定した。これまでのノウハウと研究資源を他の生命現象にも応用すべく、代謝異常を検出するMCF10A三次元培養系と、代謝関連データベース、書誌情報を統合させたローコストのゲノムワイド新規細胞制御遺伝子の探索システムを構築して、癌代謝を制御する遺伝子をスクリーニングすることを計画した。今年度はこれまでに論文報告がなされている遺伝子について、結果の追試実験を行った。まず、セリンの生合成に関わるPHGDHをMCF10A細胞に導入し、マトリゲル中での三次元培養を行った。論文上は上皮細胞の極性が失われることが報告されていたが、これを再現することはできなかった。また、GLDCもまたNIH3T3をトランスフォームすることが報告されていたが、これも我々の細胞では再現を取ることができなかった。以上の結果より、代謝に関わる酵素の過剰発現だけで癌細胞の形質を付与することが可能なのかどうか、実験系の構築から改めて考え直さなければならないと言える。

  • 初代培養乳腺上皮細胞を用いたBasal-like乳癌発癌に関与する遺伝子の探索

    2012  

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    乳癌はLuminal-like, Basal-like, ErbB2過剰発現タイプの3つのサブタイプに大別される。中でもBasal-like乳癌は癌細胞そのものの増殖能や運動性が高いことに加えて、従来の治療標的分子であるホルモン受容体やERBB2の発現が見られず有効な分子標的治療法が存在しないため予後が悪い。本研究ではこのBasal-like乳癌における発癌および癌悪性化に関する因子を網羅的に解析するため、マウス由来の初代培養乳腺上皮細胞とマトリゲル三次元培養を用いたin vitroスクリーニング系の確立を目標として研究を行った。まず最初にBasal-like乳癌のcell of originであることが報告されているLuminal前駆細胞を含むLuminal細胞画分の単離を試みた。マウス乳腺fat padを酵素処理し得られた細胞懸濁液を白血球共通抗原CD45, 赤血球抗原TER119, 内皮細胞抗原CD31で染色して血球や血管に由来する細胞を除去し、残りの上皮および繊維芽細胞を含む画分をCD24およびCD49fで展開することでCD24high, CD49flowのLuminal細胞画分をセルソーターによって分取した。更にこの画分に含まれる細胞がLuminalケラチンであるKeratin 8およびKeratin 18を発現し、BasalケラチンであるKeratin 5およびKeratin 14を発現しないことを免疫染色およびRT-PCRによって確認した。次にこのLuminal細胞をマトリゲル上に播種して乳腺構造を模倣した管腔の形成を観察したところ、播種1日後にはマトリゲル上で細胞同士が細胞塊を形成することが分かり、播種後3~4日と早い段階で中空コロニーが形成された。また中空コロニー形成後に蛍光タンパク質を発現するレトロウイルスベクターを上清培地に加えることで中空コロニーの一部の細胞に蛍光が観察され、乳腺構造を模倣した管腔環境下での遺伝子導入が可能であることが分かった。しかしながらこのコロニーは播種8日後には中心が細胞で満たされてしまい、その後縮小してしまうことが分かった。実際の解析では中空コロニー形成後に癌遺伝子候補を導入して経過を観察することで癌化や悪性化の過程を解析することを目標としているため、中空コロニーの状態を解析期間の間維持する必要がある。そこで次に播種する細胞数の最適化を検討した。細胞数を2 x 104 cells/well程度まで薄くすると播種後細胞塊の形成は見られず、中空コロニーは播種後7~10日と遅れて形成され、その後15日目まで維持されることが分かったが1 well辺りに見られる中空コロニー数が最大50個程度となり、また実験毎にコロニー形成数が大きく変化するという結果が得られた。この原因としてLuminal細胞画分には比較的少数の増殖可能なLuminal前駆細胞と大半を占める増殖能に乏しい成熟Luminal細胞が存在し、この割合がマウスの性周期によって変動していることが考えられる。また、いくつかのwellにおいては繊維芽細胞のコンタミネーションが見られ、マトリゲルの下部で活発に増殖している様子が観察された。そこで、非上皮細胞除去の際に繊維芽細胞表面に発現するCD140a抗原に対する抗体を加えて繊維芽細胞の除去を行い、さらにLuminal細胞画分をCD61抗体で染色することでCD24high, CD49flow, CD61+の増殖可能なLuminal前駆細胞を濃縮することを考えた。これまでに、以前のLuminal前駆細胞に関する報告と同程度である、Luminal細胞画分の15~30%にCD61陽性細胞が見られることが分かった。また、実際の癌遺伝子導入実験においては癌抑制因子p53の機能を抑制しなければoncogene-induced senescenceによって最終的に細胞死が誘導されることが示唆されるため、現在p53欠損マウスにおいても解析を進めており、野生型マウスと同程度のCD61陽性Luminal細胞が得られることを確認している。今後はこのCD61陽性Lumina細胞を単離してマトリゲル上での培養を検討することで長期間中空コロニーを維持可能な系を確立しBasal-like乳癌に重要な因子の解析を行う予定である。

  • NF-kBシグナル伝達経路を標的とした高悪性乳癌の治療法の確立

    2011  

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     我々は、これまでに高悪性度の乳癌細胞株では、タンパク質キナーゼNIK依存的に転写因子NFκBが恒常的に活性化し、細胞増殖に必要な役割を果たしていることを明らかにした。さらに、NFκBが乳癌幹細胞の維持に寄与していることも明らかにした。本年度は、NIK活性化機構の解析と乳癌幹細胞維持に関わるNFκB標的遺伝子の特定を進めた。前者の解析においては、プロテオーム解析によりNIKの活性化を促進する新規タンパク質を同定した。このタンパク質はNIK活性化乳癌細胞株において高く発現しており、RNAiノックダウンにて発現を抑制するとNIK恒常的活性化は抑制され、さらにNFκBの活性化も低下した。ノックダウン細胞では細胞増殖が低下し、さらに抗癌剤耐性も下がっていた。このタンパク質の発現を欠損するマウス繊維芽細胞を用いてNIK活性化を導くサイトカイン刺激(lymphotoxin β, TWEAKなど)を行うと、NIKの活性化が顕著に減弱した。従って、このタンパク質は乳癌細胞のみならず、正常細胞においてもNIK活性化を促進する重要因子であると考えられた。この分子はタンパク質のユビキチン化を制御することが知られていることから、NIKやその制御因子のユビキチン化を調節することによりNIK活性化を導いていると予想している。 一方で、乳癌幹細胞維持に関わるNFκB標的遺伝子としてNOTCHリガンドの一つであるJAG1を同定した。乳癌細胞のNFκB を遺伝子導入やサイトカイン刺激により上昇させると乳癌幹細胞の割合が増加することを見いだしていたが、JAG1の発現抑制によりこの増加は顕著に抑制された。また、NOTCH阻害薬の添加によっても同様に抑制された。乳癌組織の構成細胞である繊維芽細胞やマクロファージにサイトカイン刺激を加えてNFκBを活性化させると、乳癌細胞と同様にJAG1発現が増加した。このことから、乳癌細胞やそれを取り巻く正常細胞群のNFκBが活性化されると、JAG1の発現が増加して乳癌幹細胞の増加に寄与すると考えられた。本研究により、NIK活性化を促進する新規タンパク質やJAG1が乳癌の新規治療標的になる可能性が見いだされた。

  • NF-κBシグナル伝達経路を標的とした高悪性乳癌の治療法の確立

    2010  

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    転写因子NFκBは、様々な癌細胞において恒常的に活性化し、細胞増殖や転移・浸潤、血管新生などに関わる様々な遺伝子の発現を誘導して癌の悪性化をもたらす。従ってNFκBは抗癌剤の格好の標的であるが、NFκB阻害剤の開発は難航していること、NFκBは感染防御や骨代謝制御などの多様な生理機能を有することから、癌細胞においてNFκB恒常的活性化を導く上流のタンパク質や癌の悪性化に関わる標的遺伝子に対する阻害剤の開発が必要とされている。 これまでに我々は、高悪性のbasal-like乳癌細胞において、タンパク質キナーゼNIK依存的にNFκBが恒常的に活性化し細胞増殖に必要な役割を果たしていることを明らかにした。本年度は、特定課題研究としてbasal-like乳癌細胞におけるNFκB標的遺伝子の探索とNIK恒常的活性化機構の解析を行った。標的遺伝子探索の結果、乳癌幹細胞の維持に関わるシグナル伝達系のリガンドを同定した(本年度日本癌学会にて発表、論文準備中)。これは、NFκBの恒常的活性化が乳癌幹細胞維持に関わることを分子レベルで示した初めての成果である。さらに、癌細胞の転移・浸潤を促進する細胞骨格制御因子も同定しており、上記リガンドやこの因子がbasal-like乳癌細胞に対する新たな治療標的になる可能性がある。NIK活性化機構の解析としては、basal-like乳癌細胞ではNIK遺伝子のエピジェネティック変異によりNIK mRNAが高発現してNFκB恒常的活性化を誘導していることを明らかにした。この成果はCancer Science誌に論文発表した(Yamamoto M et al, 2010)。さらに、プロテオーム解析によりNIK活性化に関わるユビキチン関連因子も同定しており、その分子機構について詳細な解析を進めているところである。

  • ErbB2陰性乳癌細胞の増殖を制御するシグナル因子NOTCH3の機能解析

    2010  

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    我々はこれまでErbB2の発現陰性の乳がん細胞株においてNOTCH3が増殖に必要であることを報告してきた(Yamaguchi et al, Cancer Res, 68, 1881-1888, 2008)。しかし、NOTCH3がどのような仕組みで乳癌細胞の増殖を制御しているのかは不明であった。NOTCH3はリガンドとの結合後、細胞質側がγセクレターゼなどのプロテアーゼで切断を受けて、核に移行し転写制御因子として作用する。そこで、NOTCH3の機能を調べるために、NOTCH3の発現により誘導される遺伝子発現ネットワークを解析することとした。この目的のために、ErbB2陰性のBT549細胞株をもとにNOTCH3をドキシサイクリンで誘導できる細胞系を樹立した。この細胞株にドキシサイクリンでNOTCH3を誘導して、マイクロアレイ解析を行ったところ、129個の遺伝子(発現の上がるもの91個、下がるもの38個)を同定した。これらの遺伝子の機能をみると、上皮間葉移行(EMT)と代謝に関わる遺伝子群が含まれていた。特にEMT誘導に関しては、TGF-β非依存的にEMT誘導に関わるslugとSmad3の誘導されることが示唆された。(Dong et al, in preparation )。

  • SILAC法による感染初期応答ネットワークの比較解析

    2009  

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     生物はさまざまな細菌やウイルスの感染の危険に曝されている。自然免疫は、ショウジョウバエから存在する進化的に高度に保存された防御システムである。生物は病原体の感染を受けると、その構成成分(pathogen-associated molecular patterns; PAMPs)を認識するToll-like receptor(TLR)を活性化して炎症性サイトカインの産生などの宿主応答を行う。 我々を含む複数のグループはTLR4による宿主応答反応においてチロシンリン酸化が必須であることを見いだしたが、その下流におけるシグナル伝達経路の全体を捉えられていなかった。こうした包括的な解析を可能にするプロテオミクスの実験技術が近年急速に発展した。我々は、シグナル伝達の最も初期に作動するチロシンリン酸化に焦点を当て、安定同位体でタンパク質を標識するSILAC法と呼ばれる方法で、TLR4の活性化に伴ってチロシンリン酸化されるタンパク質とリン酸化のダイナミクスを解析した。その結果、TLR4の活性化によりチロシンリン酸化をうけるタンパク質(もしくはその結合蛋白質)を61種類同定することに成功した。さらにチロシンリン酸化されるタンパク質のうち、マクロファージでの機能が未知であったBCAPについてより詳細な分子生物学的解析を行い、BCAPが選択的スプライシングにより二種類のタンパク質が発現すること、このうちの一つ(分子量の大きいL型)がSyk-PI3K-PLC-γを介するNF-κBの持続的な活性化を抑制することにより、抗炎症性サイトカインであるIL-10の発現を抑制して感染応答を調節することを明らかにした。

  • 癌細胞遺伝子発現データベースを用いた新規乳癌治療標的の同定と創薬への応用

    2009  

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     細胞分裂において、複製した染色体は厳密に二つの細胞に分配される必要がある。この分配機構が破綻すると、染色体の数が変化し(異数性)、遺伝子発現のバランスが崩れたさまざまな細胞が生まれる。癌細胞において染色体分配機構が異常になると、増殖や生存に有利な細胞が出現すると考えられる。我々は、癌の悪性化に関わる治療標的遺伝子を同定する目的で、染色体分配を制御する遺伝子に着目し、その同定と機能解析を進めている。 これまでに我々は独自に作製した125種類の癌細胞株の遺伝子発現プロファイルの解析から、がんの発症と悪性化にかかわる可能性のある新たな遺伝子増幅部位を同定した。最近、このデータベースとタンパク質局在データベースなどとの複数のデータベースの相互比較により、中心体、紡錘体、キネトコアなどに局在し、染色体分配に関わると予想された機能未知の24種類の遺伝子を新たに抽出した。これらの遺伝子について作製したsiRNAをHeLa細胞にトランスフェクションし、細胞増殖能とノコダゾールによるM期停止能を調べたところ、5種の遺伝子を抑制した細胞では、いずれも対照のHeLa細胞と比較して、増殖の抑制に加えて明らかな mitotic index の増加を認め、M期の進行に異常があることが示唆された。そのうちの1種類については、タイムラプス観察によりM期通過に要する時間を測定すると正常細胞の2倍以上であり、正常な分裂ができず多核となった細胞、多極性の紡錘体が観察された。また、微小管重合阻害剤であるノコダゾールを添加して行った実験では、1種類の遺伝子について、紡錘体チェックポイント機構によるM期停止が起こらず、多核を持った間期細胞が多数観察された。 以上の結果から、これらの遺伝子は新規の染色体分配制御遺伝子であると考え、さらに詳細な解析を進めている。

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Teaching Experience

  • がんの生物学

  • 分子細胞生物学A, B, C