Researcher Profile and Settings

Affiliation

• Faculty of Medicine　Global Center for BiomedicalScience and Engineering

Job Title

• Associate Professor

URL

http://kaken.nii.ac.jp/d/r/90435561.ja.html

J-Global ID

• 201301059615076240

Research Interests

• Synthetic Metabology   Synthetic Biology   乳癌   糖代謝   Warburg効果   3次元培養   シグナル伝達   

Research Areas

• Life sciences / Molecular biology

Educational Organization

•  Bachelor's degree program　Departments of Medicine, School of Medicine
•  Master's degree program　Graduate School of Biomedical Science and Engineering
•  Doctoral (PhD) degree program　Graduate School of Biomedical Science and Engineering

Academic & Professional Experience

• 2020/12 - Today 北海道大学大学院医学研究院 医理工学グローバルセンター 准教授
• 2014/10 - Today 北海道大学 医学(系)研究科(研究院) 講師
• 2009/10 - 2014/09 北海道大学 医学(系)研究科(研究院) 助教
• 2007/10 - 2009/09 ローレンスバークレー国立研究所 博士研究員
• 2006/04 - 2007/09 大阪バイオサイエンス研究所 特別研究員

Research Activities

Published Papers

• LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity.

  Yuichi Mazaki, Haruka Handa, Yoshizuki Fumoto, Takahiro Horinouchi, Yasuhito Onodera

  Cell communication and signaling : CCS 21 (1) 300 - 300 2023/10/30 

  BACKGROUND: Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells. METHODS: Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2. RESULTS: fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP. CONCLUSIONS: LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract.
• p53 secures the normal behavior of H3.1 histone in the nucleus by regulating nuclear phosphatidic acid and EZH2 during the G1/S phase

  Tsukasa Oikawa, Junya Hasegawa, Haruka Handa, Naomi Ohnishi, Yasuhito Onodera, Ari Hashimoto, Junko Sasaki, Takehiko Sasaki, Koji Ueda, Hisataka Sabe

  2023/06/28 

  Abstract Histones are key molecules of epigenetic regulation and inheritance, and are thought to be chaperoned and transported into the nucleus appropriately prior to being integrated into nucleosomes. H3.1 histone is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, as a new component of duplicating nucleosomes. Here we found that p53 is necessary to secure the normal behavior and modification of H3.1 in the nucleus during the G1/S phase, in which p53 increases C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1) levels and decreases enhancer of zeste homolog 2 (EZH2) levels in the H3.1 interactome. In the absence of p53, H3.1 molecules tended to be tethered at or near the nuclear envelope (NE), where they were predominantly trimethylated at lysine 27 (H3K27me3) by EZH2, without forming nucleosomes. This accumulation was likely caused by the high affinity of H3.1 towards phosphatidic acid (PA). p53 reduced nuclear PA levels by increasing levels of CTDNEP1, which activates lipin to convert PA into diacylglycerol. Induction of theTMEM255Agene by p53 linked p53 with CTDNEP1, in which TMEM255A stabilized CTDNEP1. We moreover found that the cytosolic H3 chaperone HSC70 attenuates the H3.1-PA interaction, and our molecular imaging analyses suggested that H3.1 molecules may be anchored around the NE after their nuclear entry. Our results expand our knowledge of p53 function in regulation of the nuclear behavior of H3.1 during the G1/S phase, in which p53 may primarily target nuclear PA and EZH2.
• p53 controls the nuclear entry and epigenetic modification of H3.1 by downregulating nuclear phosphatidic acid

  Tsukasa Oikawa, Junya Hasegawa, Naomi Ohnishi, Yasuhito Onodera, Ari Hashimoto, Junko Sasaki, Takehiko Sasaki, Koji Ueda, Hisataka Sabe

  2023/06/28
• Angiogenic inhibitor pre-administration improves the therapeutic effects of immunotherapy.

  Mineyoshi Sato, Nako Maishi, Yasuhiro Hida, Aya Yanagawa-Matsuda, Mohammad Towfik Alam, Jun Sakakibara-Konishi, Jin-Min Nam, Yasuhito Onodera, Satoshi Konno, Kyoko Hida

  Cancer medicine 2023/02/19 

  In lung cancer, immune checkpoint inhibitors (ICIs) are often inadequate for tumor growth inhibition. Angiogenic inhibitors (AIs) are required to normalize tumor vasculature for improved immune cell infiltration. However, in clinical practice, ICIs and cytotoxic antineoplastic agents are simultaneously administered with an AI when tumor vessels are abnormal. Therefore, we examined the effects of pre-administering an AI for lung cancer immunotherapy in a mouse lung cancer model. Using DC101, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody, a murine subcutaneous Lewis lung cancer (LLC) model was used to determine the timing of vascular normalization. Microvessel density (MVD), pericyte coverage, tissue hypoxia, and CD8-positive cell infiltration were analyzed. The effects of an ICI and paclitaxel after DC101 pre-administration were investigated. On Day 3, increased pericyte coverage and alleviated tumor hypoxia represented the highest vascular normalization. CD8+ T-cell infiltration was also highest on Day 3. When combined with an ICI, DC101 pre-administration significantly reduced PD-L1 expression. When combined with an ICI and paclitaxel, only DC101 pre-administration significantly inhibited tumor growth, but simultaneous administration did not. AI pre-administration, and not simultaneous administration, may increase the therapeutic effects of ICIs due to improved immune cell infiltration.
• The oral bacterium Streptococcus mutans promotes tumor metastasis by inducing vascular inflammation

  Li Yu, Nako Maishi, Erika Akahori, Akira Hasebe, Ryo Takeda, Aya Yanagawa Matsuda, Yasuhiro Hida, Jin‐Min Nam, Yasuhito Onodera, Yoshimasa Kitagawa, Kyoko Hida

  Cancer Science 1347-9032 2022/08/23 [Refereed]
  
• The oxidized‐LDL/LOX‐1 axis in tumor endothelial cells enhances metastasis by recruiting neutrophils and cancer cells

  Takuya Tsumita, Nako Maishi, Dorcas Akuba‐Muhyia Annan, Mohammad Alam Towfik, Aya Matsuda, Yasuhito Onodera, Jin‐Min Nam, Yasuhiro Hida, Kyoko Hida

  International Journal of Cancer 0020-7136 2022/06/09
• A Consistent Protocol Reveals a Large Heterogeneity in the Biological Effectiveness of Proton and Carbon-Ion Beams for Various Sarcoma and Normal-Tissue-Derived Cell Lines.

  Masashi Yagi, Yutaka Takahashi, Kazumasa Minami, Taeko Matsuura, Jin-Min Nam, Yasuhito Onodera, Takashi Akagi, Takuya Maeda, Tomoaki Okimoto, Hiroki Shirato, Kazuhiko Ogawa

  Cancers 14 (8) 2022/04/15 

  This study investigated variations in the relative biological effectiveness (RBE) values among various sarcoma and normal-tissue-derived cell lines (normal cell line) in proton beam and carbon-ion irradiations. We used a consistent protocol that specified the timing of irradiation after plating cells and detailed the colony formation assay. We examined the cell type dependence of RBE for proton beam and carbon-ion irradiations using four human sarcoma cell lines (MG63 osteosarcoma, HT1080 fibrosarcoma, SW872 liposarcoma, and SW1353 chondrosarcoma) and three normal cell lines (HDF human dermal fibroblast, hTERT-HME1 mammary gland, and NuLi-1 bronchus epithelium). The cells were irradiated with gamma rays, proton beams at the center of the spread-out Bragg peak, or carbon-ion beams at 54.4 keV/μm linear energy transfer. In all sarcoma and normal cell lines, the average RBE values in proton beam and carbon-ion irradiations were 1.08 ± 0.11 and 2.08 ± 0.36, which were consistent with the values of 1.1 and 2.13 used in current treatment planning systems, respectively. Up to 34% difference in the RBE of the proton beam was observed between MG63 and HT1080. Similarly, a 32% difference in the RBE of the carbon-ion beam was observed between SW872 and the other sarcoma cell lines. In proton beam irradiation, normal cell lines had less variation in RBE values (within 10%), whereas in carbon-ion irradiation, RBE values differed by up to 48% between hTERT-HME1 and NuLi-1. Our results suggest that specific dose evaluations for tumor and normal tissues are necessary for treatment planning in both proton and carbon-ion therapies.
• Proteolysis of a histone acetyl reader, ATAD2, induces chemoresistance of cancer cells under severe hypoxia by inhibiting cell cycle progression in S phase

  Takao Haitani, Minoru Kobayashi, Sho Koyasu, Shusuke Akamatsu, Tatsuya Suwa, Yasuhito Onodera, Jin-Min Nam, Phuong Thi Lien Nguyen, Toshi Menju, Hiroshi Date, Osamu Ogawa, Hiroshi Harada

  Cancer Letters 528 76 - 84 0304-3835 2022/03 [Refereed]
  
• Roles of MFN2 and MFN2 associated protein in chemotaxis of neutrophil-like differentiated HL-60 cells

  Mazaki Yuichi, Higashi Tsunehito, Nio-Kobayashi Junko, Onodera Yasuhito

  Proceedings for Annual Meeting of The Japanese Pharmacological Society 公益社団法人 日本薬理学会 96 2-B-P-112  2022 

  Neutrophils are important in innate immunity and in the initiation of an acute response to infection. Under normal conditions, the mitochondrial membrane potential of neutrophils is low, and neutrophils energy depends fundamentally on glycolysis. In contrast, neutrophils require energy supplied from mitochondrial oxidative phosphorylation (OXPHOS) during infection. The inhibition of mitochondrial OXPHOS blocks the chemotaxis of neutrophils. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. Furthermore, the silencing of MFN2 associated protein suppresses also mitochondrial morphological changes and chemotaxis upon fMLP stimulation. These results suggest that MFN2 and MFN2 associated protein are involved in chemotaxis of differentiated HL-60 cells.
• Dual roles of AMAP1 in the transcriptional regulation and intracellular trafficking of carbonic anhydrase IX

  Mei Horikawa, Hisataka Sabe, Yasuhito Onodera

  Translational Oncology 15 (1) 101258 - 101258 1936-5233 2022/01 [Refereed][Not invited]
   

  BACKGROUND: The cell-surface enzyme carbonic anhydrase IX (CAIX/CA9) promotes tumor growth, survival, invasion, and metastasis, mainly via its pH-regulating functions. Owing to its tumor-specific expression, CAIX-targeting antibodies/chemicals are utilized for therapeutic and diagnostic purposes. However, mechanisms of CAIX trafficking, which affects such CAIX-targeting modalities remain unclear. In this study, roles of the AMAP1-PRKD2 pathway, which mediates integrin recycling of invasive cancer cells, in CAIX trafficking were investigated. METHODS: Using highly invasive MDA-MB-231 breast cancer cells, the physical association and colocalization of endogenous proteins were analyzed by immunoprecipitation and immunofluorescence, protein/mRNA levels were quantified by western blotting/qPCR, and cell-surface transport and intracellular/extracellular pH regulation were measured by biotin-labeling and fluorescent dye-based assays, respectively. The correlation between mRNA levels and patients' prognoses was analyzed using a TCGA breast cancer dataset. RESULTS: AMAP1 associated with the CAIX protein complex, and they colocalized at the plasma membrane and tubulovesicular structures. AMAP1 knockdown reduced total/surface CAIX, induced its lysosomal accumulation and degradation, and affected intracellular/extracellular pH. PRKD2 knockdown excluded AMAP1 from the CAIX complex and reduced total CAIX in a lysosome-dependent manner. Unexpectedly, AMAP1 knockdown also reduced CAIX mRNA. AMAP1 interacted with PIAS3, which stabilizes HIF-1α, a transcriptional regulator of CA9. AMAP1 knockdown inhibited the PIAS3-HIF-1α interaction and destabilized the HIF-1α protein. High-ASAP1 (AMAP1-encoding gene) together with high-PIAS3 correlated with high-CA9 and an unfavorable prognosis in breast cancer. CONCLUSION: The AMAP1-PRKD2 pathway regulates CAIX trafficking, and modulates its total/surface expression. The AMAP1-PIAS3 interaction augments CA9 transcription by stabilizing HIF-1α, presumably contributing to an unfavorable prognosis.
• Strategies for all-at-once and stepwise selection of cells with multiple genetic manipulations

  Mei Horikawa, Hisataka Sabe, Yasuhito Onodera

  Biochemical and Biophysical Research Communications 582 93 - 99 0006-291X 2021/10 [Refereed][Not invited]
   

  The genetic manipulation of cells followed by their selection is indispensable for cell biological research. Although antibiotics-resistant genes are commonly used as selection markers, optimization of the condition for each selective agent is required. Here we utilized split-inteins and the drug-selectable marker puromycin N-acetyltransferase (PAC) to develop a system that enables the selection of cells simultaneously or sequentially transfected with multiple genetic constructs, using only puromycin. The active PAC enzyme was reconstituted by intein-mediated trans-splicing at several inherent or engineered serine/cysteine residues. Multiple splitting and reconstitution of active PAC was readily achieved by selecting optimum division sites based on the cellular tolerance to various puromycin concentrations. To achieve the stepwise selection method, PAC-intein fragments were transduced into cells using a virus-like particle (VLP) composed of HIV-1 gag-pol and VSV-G. The PAC-intein-VLP successfully conferred sufficient PAC activity for puromycin selection, which was quickly diminished in the absence of the VLP. Our findings demonstrate a versatile strategy for establishing markers for all-at-once or stepwise selection of multiple genetic manipulations, which will be useful in many fields of biology.
• Lysosomal trafficking mediated by Arl8b and BORC promotes invasion of cancer cells that survive radiation

  Ping-Hsiu Wu, Yasuhito Onodera, Amato J. Giaccia, Quynh-Thu Le, Shinichi Shimizu, Hiroki Shirato, Jin-Min Nam

  Communications Biology 3 (1) 2020/12 [Refereed][Not invited]
   

  Enhanced invasiveness, a critical determinant of metastasis and poor prognosis, has been observed in cancer cells that survive cancer therapy, including radiotherapy. Here, we show that invasiveness in radiation-surviving cancer cells is associated with alterations in lysosomal exocytosis caused by the enhanced activation of Arl8b, a small GTPase that regulates lysosomal trafficking. The binding of Arl8b with its effector, SKIP, is increased after radiation through regulation of BORC-subunits. Knockdown of Arl8b or BORC-subunits decreases lysosomal exocytosis and the invasiveness of radiation-surviving cells. Notably, high expression ofARL8Band BORC-subunit genes is significantly correlated with poor prognosis in breast cancer patients. Sp1, an ATM-regulated transcription factor, is found to increase BORC-subunit genes expression after radiation. In vivo experiments show that ablation of Arl8b decreases IR-induced invasive tumor growth and distant metastasis. These findings suggest that BORC-Arl8b-mediated lysosomal trafficking is a target for improving radiotherapy by inhibiting invasive tumor growth and metastasis.
• 核ラミナにおいてp53はEZH2を妨害してH3K27高メチル化を阻害する(p53 counteracts EZH2 at the nuclear lamina to prevent H3K27 hypermethylation)

  及川 司, 大西 なおみ, 小野寺 康仁, 橋本 あり, 植田 幸嗣, 佐邊 壽孝

  日本生化学会大会プログラム・講演要旨集 93回 [P - 466] 2020/09
• Rab27b contributes to radioresistance and exerts a paracrine effect via epiregulin in glioblastoma

  Soichiro Nishioka, Ping-Hsiu Wu, Toshiaki Yakabe, Amato J Giaccia, Quynh-Thu Le, Hidefumi Aoyama, Shinichi Shimizu, Hiroki Shirato, Yasuhito Onodera, Jin-Min Nam

  Neuro-Oncology Advances 2 (1) 2020/01/01 [Refereed][Not invited]
   

  Radiotherapy is the standard treatment for glioblastoma (GBM). However, radioresistance of GBM cells leads to recurrence and poor patient prognosis. Recent studies suggest that secretion factors have important roles in radioresistance of tumor cells. This study aims to determine whether Rab27b, a small GTPase involved in secretory vesicle trafficking, plays a role in radioresistance of GBM. Microarray analysis, cell viability analysis, apoptosis assay, immunostaining, and in vivo experiments were performed to assess the effect of Rab27b on radioresistance of GBM. We further investigated paracrine effects mediated by Rab27b after X-ray irradiation using coculture systems of glioma cell lines. Rab27b was specifically upregulated in irradiated U87MG cells. Furthermore, Rab27b knockdown decreased the proliferation of GBM cells after irradiation. Knockdown of Rab27b in U87MG cells combined with radiation treatment suppressed orthotopic tumor growth in the mouse brain and prolonged the survival of recipient mice. Interestingly, the co-upregulation of Rab27b and epiregulin (EREG), a member of the epidermal growth factor (EGF) family, correlated with radioresistance in glioma cell lines. Additionally, EREG, which was secreted from U87MG cells via Rab27b-mediated mechanism, activated EGF receptor and contributed to H4 cell proliferation in a paracrine manner. Our results show that Rab27b mediates the radioresistance of highly malignant GBM cells. Rab27b promotes the proliferation of adjacent cells through EREG-mediated paracrine signaling after irradiation. Thus, the Rab27b-EREG pathway is a novel potential target to improve the efficacy of radiotherapy in GBM.
• Targeting Integrins in Cancer Nanomedicine: Applications in Cancer Diagnosis and Therapy

  Ping-Hsiu Wu, Abayomi Emmanuel Opadele, Yasuhito Onodera, Jin-Min Nam

  Cancers 11 (11) 1783 - 1783 2019/11/13 

  Due to advancements in nanotechnology, the application of nanosized materials (nanomaterials) in cancer diagnostics and therapeutics has become a leading area in cancer research. The decoration of nanomaterial surfaces with biological ligands is a major strategy for directing the actions of nanomaterials specifically to cancer cells. These ligands can bind to specific receptors on the cell surface and enable nanomaterials to actively target cancer cells. Integrins are one of the cell surface receptors that regulate the communication between cells and their microenvironment. Several integrins are overexpressed in many types of cancer cells and the tumor microvasculature and function in the mediation of various cellular events. Therefore, the surface modification of nanomaterials with integrin-specific ligands not only increases their binding affinity to cancer cells but also enhances the cellular uptake of nanomaterials through the intracellular trafficking of integrins. Moreover, the integrin-specific ligands themselves interfere with cancer migration and invasion by interacting with integrins, and this finding provides a novel direction for new treatment approaches in cancer nanomedicine. This article reviews the integrin-specific ligands that have been used in cancer nanomedicine and provides an overview of the recent progress in cancer diagnostics and therapeutic strategies involving the use of integrin-targeted nanomaterials.
• DNA複製中の核内におけるK27トリメチル化ヒストンH3の挙動に対するp53の必要性(Requirement for p53 in intra-nuclear dynamics of the K27-trimethylated histone H3 during DNA replication)

  及川 司, 大西 なおみ, 小野寺 康仁, 橋本 あり, 植田 幸嗣, 佐邊 壽孝

  日本生化学会大会プログラム・講演要旨集 92回 [2T13m - 01] 2019/09
• Lambda-Carrageenan Enhances the Effects of Radiation Therapy in Cancer Treatment by Suppressing Cancer Cell Invasion and Metastasis through Racgap1 Inhibition

  Ping-Hsiu Wu, Yasuhito Onodera, Frances C. Recuenco, Amato J. Giaccia, Quynh-Thu Le, Shinichi Shimizu, Hiroki Shirato, Jin-Min Nam

  Cancers 11 (8) 1192 - 1192 2019/08/16 

  Radiotherapy is used extensively in cancer treatment, but radioresistance and the metastatic potential of cancer cells that survive radiation remain critical issues. There is a need for novel treatments to improve radiotherapy. Here, we evaluated the therapeutic benefit of λ-carrageenan (CGN) to enhance the efficacy of radiation treatment and investigated the underlying molecular mechanism. CGN treatment decreased viability in irradiated cancer cells and enhanced reactive oxygen species accumulation, apoptosis, and polyploid formation. Additionally, CGN suppressed radiation-induced chemoinvasion and invasive growth in 3D lrECM culture. We also screened target molecules using a gene expression microarray analysis and focused on Rac GTPase-activating protein 1 (RacGAP1). Protein expression of RacGAP1 was upregulated in several cancer cell lines after radiation, which was significantly suppressed by CGN treatment. Knockdown of RacGAP1 decreased cell viability and invasiveness after radiation. Overexpression of RacGAP1 partially rescued CGN cytotoxicity. In a mouse xenograft model, local irradiation followed by CGN treatment significantly decreased tumor growth and lung metastasis compared to either treatment alone. Taken together, these results suggest that CGN may enhance the effectiveness of radiation in cancer therapy by decreasing cancer cell viability and suppressing both radiation-induced invasive activity and distal metastasis through downregulating RacGAP1 expression.
• ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer.

  Hashimoto S, Furukawa S, Hashimoto A, Tsutaho A, Fukao A, Sakamura Y, Parajuli G, Onodera Y, Otsuka Y, Handa H, Oikawa T, Hata S, Nishikawa Y, Mizukami Y, Kodama Y, Murakami M, Fujiwara T, Hirano S, Sabe H

  Proceedings of the National Academy of Sciences of the United States of America 116 (35) 17450 - 17459 0027-8424 2019/08 [Refereed][Not invited]
   

  Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.
• 乳癌における腫瘍関連MUC1の発現変化とその臨床的意義

  清水 亜衣, 畑中 佳奈子, 畑中 豊, 南家 綾江, 奥村 麻美, 燕 果歩, 成地 健太郎, 佐藤 正治, 加瀬 廣, 小野寺 康仁, 三橋 智子, 山下 啓子, 松野 吉宏

  日本乳癌学会総会プログラム抄録集 (一社)日本乳癌学会 27回 593 - 593 2019/07
• 乳癌の進展過程における腫瘍関連MUC1の発現変化

  清水 亜衣, 畑中 佳奈子, 畑中 豊, 南家 綾江, 奥村 麻美, 燕 果歩, 小野寺 康仁, 三橋 智子, 山下 啓子, 松野 吉宏

  日本病理学会会誌 (一社)日本病理学会 108 (1) 393 - 393 0300-9181 2019/04
• Mitofusin 2 is involved in chemotaxis of neutrophil-like differentiated HL-60 cells.

  Mazaki Y, Takada S, Nio-Kobayashi J, Maekawa S, Higashi T, Onodera Y, Sabe H

  Biochemical and biophysical research communications 513 (3) 708 - 713 0006-291X 2019/04 [Refereed][Not invited]
   

  Neutrophils rapidly migrate to infection sites after the recognition of invaders. During chemotaxis, neutrophils require energy supplied by mitochondria oxidative phosphorylation (OXPHOS), whereas neutrophils rely heavily on glycolysis under normal conditions. Mitochondrial OXPHOS correlates with mitochondrial morphology. Here, we examined the mitochondrial morphology of neutrophil-like differentiated HL-60 cells after chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation. We found that mitochondrial morphology changes to a tubular form after fMLP stimulation. Mitochondrial OXPHOS activity and mitochondrial complex II significantly increased after fMLP stimulation. On the other hand, the silencing of mitochondrial fusion protein mitofusin 2 (MFN2) suppresses mitochondrial morphological changes. Furthermore, MFN2 silencing suppressed OXPHOS activation and chemotaxis after fMLP stimulation. These results suggest that MFN2 is involved in chemotaxis of differentiated HL-60 cells depending on mitochondria.
• Endothelin type B receptor interacts with the 78-kDa glucose-regulated protein.

  Yuichi Mazaki, Shingo Takada, Junko Nio-Kobayashi, Satoshi Maekawa, Tsunehito Higashi, Yasuhito Onodera, Hisataka Sabe

  FEBS letters Wiley 593 (6) 644 - 651 0014-5793 2019/03 [Refereed][Not invited]
   

  Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization.
• GRP78 promotes ERK activation through endothelin type B receptor

  Mazaki Yuichi, Higashi Tsunehito, Onodera Yasuhito, Nam Jin-Min, Hashimoto Ari, Hashimoto Shigeru, Horinouchi Takahiro, Miwa Soichi

  Proceedings for Annual Meeting of The Japanese Pharmacological Society 公益社団法人 日本薬理学会 92 1-P-110  2019 [Not refereed]
   

  Endothelin (ET)-1 is involved in various diseases, including cancer, hypertension, atherosclerosis, diabetes, and fibrotic diseases, although ET-1 is originally identified as endothelium-derived vasocontractile peptide. ET receptors belong to the class A of G protein-coupled receptor, and consist of ET type A receptor (ET_AR) and ET type B receptor (ET_BR). ET_AR and ET_BR generally exhibit the opposite responses, although many exceptions exist. Here, we attempted to identify ET_AR or ET_BR specific binding proteins to understand difference of ET_AR- and ET_BR-mediated responses upon ET-1 stimulation. We found that GRP78 exhibited a stronger binding affinity toward ET_BR than ET_AR. Overexpression of GRP78 promotes ET_BR-mediated ERK activation. In addition, the silencing of GRP78 suppressed ET_BR-mediated ERK activation. On the other hand, ET_BR can localize GRP78 to cell periphery. Our results suggest that interaction of ET_BR with GRP78 affects the ERK activation and GRP78 localization.
• Arf6-driven cell invasion is intrinsically linked to TRAK1-mediated mitochondrial anterograde trafficking to avoid oxidative catastrophe.

  Onodera Y, Nam JM, Horikawa M, Shirato H, Sabe H

  Nature Communications 9 (1) 2682 - 2682 2018/07 [Refereed][Not invited]
   

  Mitochondria dynamically alter their subcellular localization during cell movement, although the underlying mechanisms remain largely elusive. The small GTPase Arf6 and its signaling pathway involving AMAP1 promote cell invasion via integrin recycling. Here we show that the Arf6-AMAP1 pathway promote the anterograde trafficking of mitochondria. Blocking the Arf6-based pathway causes mitochondrial aggregation near the microtubule-organizing center, and subsequently induces detrimental reactive oxygen species (ROS) production, likely via a mitochondrial ROS-induced ROS release-like mechanism. The Arf6-based pathway promotes the localization of ILK to focal adhesions to block RhoT1-TRAK2 association, which controls mitochondrial retrograde trafficking. Blockade of the RhoT1-TRAK1 machinery, rather than RhoT1-TRAK2, impairs cell invasion, but not two-dimensional random cell migration. Weakly or non-invasive cells do not notably express TRAK proteins, whereas they clearly express their mRNAs. Our results identified a novel association between cell movement and mitochondrial dynamics, which is specific to invasion and is necessary for avoiding detrimental ROS production.
• Necessity of p53-binding to the CDH1 locus for its expression defines two epithelial cell types differing in their integrity.

  Oikawa T, Otsuka Y, Onodera Y, Horikawa M, Handa H, Hashimoto S, Suzuki Y, Sabe H

  Scientific Reports 8 (1) 1595 - 1595 2018/01 [Refereed][Not invited]
   

  TP53 mutation (i.e., loss of normal-p53) may evoke epithelial-mesenchymal transition (EMT), which was previously attributed to loss of certain miRNAs. However, not all epithelial cells undergo EMT upon TP53 mutation, and the p53-miRNA axis may not fully explain p53 function in epithelial integrity. We here show two modes of epithelial integrity: one involves p53-binding to a nucleotide region and the other does not. In the former, p53 binds to the CDH1 (encoding E-cadherin) locus to antagonize EZH2-mediated H3K27 trimethylation (H3K27me3) to maintain high levels of acetylation of H3K27 (H3K27ac). In the latter, the same locus is not highly acetylated at H3K27, and does not allow p53-binding, nor needs to antagonize EZH2. We moreover demonstrated that although the CDH1 locus in the p53-independent cells, but not in fibroblasts, becomes high-H3K27ac by butyrate and allows p53-biniding, their CDH1 expression does not become dependent on p53. Our results identified novel modes of the epithelial integrity, in which the same epithelial-specific gene locus exhibits different requirement for p53 with different histone modifications among different epithelial cells to warrant its expression.
• ARF1 recruits RAC1 to leading edge in neutrophil chemotaxis

  Yuichi Mazaki, Yasuhito Onodera, Tsunehito Higashi, Takahiro Horinouchi, Tsukasa Oikawa, Hisataka Sabe

  CELL COMMUNICATION AND SIGNALING 15 (1) 36  1478-811X 2017/10 [Refereed][Not invited]
   

  Background: The small GTPase ARF1 mediates membrane trafficking mostly from the Golgi, and is essential for the G protein-coupled receptor (GPCR)-mediated chemotaxis of neutrophils. In this process, ARF1 is activated by the guanine nucleotide exchanger GBF1, and is inactivated by the GTPase-activating protein GIT2. Neutrophils generate the G beta gamma.-PAK1-aPIX-GIT2 linear complex during GPCR-induced chemotaxis, in which aPIX activates RAC1/CDC42, which then employs PAK1. However, it has remained unclear as to why GIT2 is included in this complex. Results: We investigated the association between ARF1 and RAC1/CDC42 during the fMLP-stimulated chemotaxis of HL60 cells. We found that the silencing of GBF1 significantly impaired the recruitment of RAC1 to the leading edges, but not PAK1, aPIX, RAC2, or CDC42. A significant population of RAC1 colocalized with ARF1 at the leading edges in stimulated cells, whereas fMLP activated both ARF1 and ARF5. Consistently, the silencing of ARF1, but not ARF5, impaired the recruitment of RAC1, whereas the silencing of RAC1 did not affect the recruitment of ARF1 to the leading edges. Conclusions: Our results indicated that the activation of ARF1 triggers the plasma membrane recruitment of RAC1 in GPCR-mediated chemotaxis, which is essential for cortical actin remodeling. Thus, membrane remodeling at the leading edges appears to precede actin remodeling in chemotaxis. Together with the fact that GIT2, which inactivates ARF1, is an integral component of the machinery activating RAC1, we proposed a model in which the ARF1-RAC1 linkage enables the regulation of ARF1 by repetitive on/off cycles during GPCR-mediated neutrophil chemotaxis.
• がん代謝にもとづく生物像の理解(代謝ネットワーク) インテグリンシグナルとがん代謝

  小野寺 康仁, 佐邊 壽孝

  日本癌学会総会記事 76回 S3 - 4 0546-0476 2017/09
• Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes

  Shunsuke Kon, Kojiro Ishibashi, Hiroto Katoh, Sho Kitamoto, Takanobu Shirai, Shinya Tanaka, Mihoko Kajita, Susumu Ishikawa, Hajime Yamauchi, Yuta Yako, Tomoko Kamasaki, Tomohiro Matsumoto, Hirotaka Watanabe, Riku Egami, Ayana Sasaki, Atsuko Nishikawa, Ikumi Kameda, Takeshi Maruyama, Rika Narumi, Tomoko Morita, Yoshiteru Sasaki, Ryosuke Enoki, Sato Honma, Hiromi Imamura, Masanobu Oshima, Tomoyoshi Soga, Jun-ichi Miyazaki, Michael R. Duchen, Jin-Min Nam, Yasuhito Onodera, Shingo Yoshioka, Junichi Kikuta, Masaru Ishii, Masamichi Imajo, Eisuke Nishida, Yoichiro Fujioka, Yusuke Ohba, Toshiro Sato, Yasuyuki Fujita

  NATURE CELL BIOLOGY 19 (5) 530 - + 1465-7392 2017/05 [Refereed][Not invited]
   

  Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
• Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

  Ping-Hsiu Wu, Yasuhito Onodera, Yuki Ichikawa, Erinn B. Rankin, Amato J. Giaccia, Yuko Watanabe, Wei Qian, Takayuki Hashimoto, Hiroki Shirato, Jin-Min Nam

  INTERNATIONAL JOURNAL OF NANOMEDICINE 12 5069 - 5085 1178-2013 2017 [Refereed][Not invited]
   

  Gold nanoparticles (AuNPs) have recently attracted attention as clinical agents for enhancing the effect of radiotherapy in various cancers. Although radiotherapy is a standard treatment for cancers, invasive recurrence and metastasis are significant clinical problems. Several studies have suggested that radiation promotes the invasion of cancer cells by activating molecular mechanisms involving integrin and fibronectin (FN). In this study, polyethylene-glycolylated AuNPs (P-AuNPs) were conjugated with Arg-Gly-Asp (RGD) peptides (RGD/P-AuNPs) to target cancer cells expressing RGD-binding integrins such as alpha 5-and alpha v-integrins. RGD/P-AuNPs were internalized more efficiently and colocalized with integrins in the late endosomes and lysosomes of MDA-MB-231 cells. A combination of RGD/P-AuNPs and radiation reduced cancer cell viability and increased DNA damage compared to radiation alone in MDA-MB-231 cells. Moreover, the invasive activity of breast cancer cell lines after radiation treatment was significantly inhibited in the presence of RGD/P-AuNPs. Microarray analyses revealed that the expression of FN in irradiated cells was suppressed by combined use of RGD/P-AuNPs. Reduction of FN and downstream signaling may be involved in suppressing radiation-induced invasive activity by RGD/P-AuNPs. Our study suggests that RGD/P-AuNPs can target integrin-overexpressing cancer cells to improve radiation therapy by suppressing invasive activity in addition to sensitization. Thus, these findings provide a possible clinical strategy for using AuNPs to treat invasive breast cancer following radiotherapy.
• High expression of EPB41L5, an integral component of the Arf6-driven mesenchymal program, correlates with poor prognosis of squamous cell carcinoma of the tongue

  Yutaro Otsuka, Hiroki Sato, Tsukasa Oikawa, Yasuhito Onodera, Jin-Min Nam, Ari Hashimoto, Kiyoshi Fukunaga, Kanako C. Hatanaka, Yutaka Hatanaka, Yoshihiro Matsuno, Satoshi Fukuda, Hisataka Sabe

  CELL COMMUNICATION AND SIGNALING 14 (1) 28  1478-811X 2016/11 [Refereed][Not invited]
   

  Background: Squamous cell carcinoma of the tongue (tongue SCC) is a major subtype of head and neck squamous cell carcinoma (HNSCC), which is an intractable cancer under current therapeutics. ARF6 and its effector AMAP1 are often overexpressed in different types of cancers, such as breast cancer and renal cancer, and in these cancers, AMAP1 binds to EPB41L5 to promote invasion, metastasis, and drug resistance. EPB41L5 is a mesenchymal-specific protein, normally induced during epithelial-mesenchymal transition (EMT) to promote focal adhesion dynamics. Similarly to breast cancer and renal cancer, the acquisition of mesenchymal phenotypes is the key process that drives the malignancy of HNSCC. We previously showed that the overexpression of AMAP1 in tongue SCC is statistically correlated with the poor outcome of patients. In this study, we examined whether tongue SCC also expresses EPB41L5 at high levels. Results: Immunohistochemical staining of clinical specimens of tongue SCC demonstrated that high expression levels of EPB41L5 statistically correlate with poor disease-free survival and poor overall survival rates of patients. The tongue SCC cell line SCC-9, which overexpress Arf6 and AMAP1, also expressed EPB41L5 at high levels to promote invasiveness, whereas the weakly invasive SCC-25 cells did not express EPB41L5 at notable levels. Among the different EMT-associated transcriptional factors, ZEB1 was previously found to be most crucial in inducing EPB41L5 in breast cancer and renal cancer. In contrast, expression levels of ZEB1 did not correlate with the expression levels of EPB41L5 in tongue SCC, whereas KLF8 and FOXO3 levels showed positive correlations with EPB41L5 levels. Moreover, silencing of EPB41L5 only marginally improved the drug resistance of SCC-9 cells, even when coupled with ionizing radiation. Conclusion: Our results indicate that activation of the cancer mesenchymal program in tongue SCC, which leads to EPB41L5 expression, closely correlates with the poor prognosis of patients. However, ZEB1 was not the major inducer of EPB41L5 in tongue SCC, unlike in breast cancer and renal cancer. Thus, processes that trigger the mesenchymal program of tongue SCC, which drives their malignancies, seem to be substantially different from those of other cancers.
• ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance

  A. Hashimoto, S. Hashimoto, H. Sugino, A. Yoshikawa, Y. Onodera, H. Handa, T. Oikawa, H. Sabe

  ONCOGENESIS 5 (9) e259  2157-9024 2016/09 [Refereed][Not invited]
   

  Onset of the cancer mesenchymal program is closely associated with cancer malignancy and drug resistance. Among the different epithelial-mesenchymal transition (EMT)-associated transcriptional factors, ZEB1 has a key role in inducing the mesenchymal phenotypes and stem cell-like properties of different breast cancer cells. ARF6 and its effector AMAP1 are frequently overexpressed in breast cancer cells, and promote invasion, metastasis and drug resistance. EPB41L5 is induced during EMT, and mediates the disruption of E-cadherin-based cell-cell adhesion and the promotion of focal adhesion dynamics. Here we show that EPB41L5 is an integral component of the ARF6-based pathway, which is induced by ZEB1. We found that EPB41L5 is expressed at high levels in malignant breast cancer cells and binds to AMAP1. ZEB1 induced EPB41L5 both in cancer cells and normal cells. This relationship was recaptured with The Cancer Genome Atlas RNASeq data set, and correlated with the poor outcome of the patients. In contrast, diversified events, such as tumor growth factor beta 1 stimulation, expression of SNAI1 and TP53 mutation, can each cause the induction of ZEB1 and EPB41L5, depending on the cellular context. Our results demonstrated that the ZEB1-EPB41L5 axis is at the core of the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance of significant populations of primary breast cancers, and is tightly correlated with the poor outcomes of patients.
• The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial-mesenchymal transition

  Y. Komiya, Y. Onodera, M. Kuroiwa, S. Nomimura, Y. Kubo, J-M Nam, K. Kajiwara, S. Nada, C. Oneyama, H. Sabe, M. Okada

  ONCOGENESIS 5 (9) e258  2157-9024 2016/09 [Refereed][Not invited]
   

  Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial-mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-beta-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-a or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT.
• P53-and mevalonate pathway-driven malignancies require Arf6 for metastasis and drug resistance

  Ari Hashimoto, Tsukasa Oikawa, Shigeru Hashimoto, Hirokazu Sugino, Ayumu Yoshikawa, Yutaro Otsuka, Haruka Handa, Yasuhito Onodera, Jin-Min Nam, Chitose Oneyama, Masato Okada, Mitsunori Fukuda, Hisataka Sabe

  JOURNAL OF CELL BIOLOGY 213 (1) 81 - 95 0021-9525 2016/04 [Refereed][Not invited]
   

  Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.
• Lysophosphatidic acid activates Arf6 to promote the mesenchymal malignancy of renal cancer

  Shigeru Hashimoto, Shuji Mikami, Hirokazu Sugino, Ayumu Yoshikawa, Ari Hashimoto, Yasuhito Onodera, Shotaro Furukawa, Haruka Handa, Tsukasa Oikawa, Yasunori Okada, Mototsugu Oya, Hisataka Sabe

  NATURE COMMUNICATIONS 7 10656  2041-1723 2016/02 [Refereed][Not invited]
   

  Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial-mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-G alpha 12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients.
• Post-transcriptional regulation and metabolism

  Yasuhito Onodera

  Seikagaku 88 (3) 308 - 313 2189-0544 2016 [Refereed][Invited]
  
• Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways

  Yasuhito Onodera, Jin-Min Nam, Mina J. Bissell

  JOURNAL OF CLINICAL INVESTIGATION 124 (1) 367 - 384 0021-9738 2014/01 [Refereed][Not invited]
   

  There is a considerable resurgence of interest in the role of aerobic glycolysis in cancer; however, increased glycolysis is frequently viewed as a consequence of oncogenic events that drive malignant cell growth and survival. Here we provide evidence that increased glycolytic activation itself can be an oncogenic event in a physiologically relevant 3D culture model. Overexpression of glucose transporter type 3 (GLUT3) in nonmalignant human breast cells activated known oncogenic signaling pathways, including EGFR, beta 1 integrin, MEK, and AKT, leading to loss of tissue polarity and increased growth. Conversely, reduction of glucose uptake in malignant cells promoted the formation of organized and growth-arrested structures with basal polarity, and suppressed oncogenic pathways. Unexpectedly and importantly, we found that unlike reported literature, in 3D the differences between "normal" and malignant phenotypes could not be explained by HIF-l alpha/2 alpha, AMPK, or mTOR pathways. Loss of epithelial integrity involved activation of RAP1 via exchange protein directly activated by cAMP (EPAC), involving also O-linked N-acetylglucosamine modification downstream of the hexosamine biosynthetic pathway. The former, in turn, was mediated by pyruvate kinase M2 (PKM2) interaction with soluble adenylyl cyclase. Our findings show that increased glucose uptake activates known oncogenic pathways to induce malignant phenotype, and provide possible targets for diagnosis and therapeutics.
• Intracellular trafficking of integrins in cancer cells

  Yasuhito Onodera, Jin-Min Nam, Hisataka Sabe

  PHARMACOLOGY & THERAPEUTICS 140 (1) 1 - 9 0163-7258 2013/10 [Refereed][Not invited]
   

  Integrins are heterodimeric cell surface receptors, which principally mediate the interaction between cells and their extracellular microenvironments. Because of their pivotal roles in cancer proliferation, survival, invasion and metastasis, integrins have been recognized as promising targets for cancer treatment. As is the case with other receptors, the localization of integrins on the cell surface has provided opportunities to block their functions by various inhibitory monoclonal antibodies. A number of small molecule agents blocking integrin-ligand binding have also been established, and some such agents are currently on the market or in clinical trials for some diseases including cancer. This review exclusively focuses on another strategy for cancer therapy, which comes from the obligate localization of integrins on the cell surface; targeting the intracellular trafficking of integrins. A number of studies have shown the essential roles of integrin trafficking in hallmarks of cancer, such as activation of oncogenic signaling pathways as well as acquisition of invasiveness. Recent findings have shown that increased integrin recycling activity is associated with some types of gain-of-function mutations of p53, a common feature of diverse types of cancers, which also indicates that targeting integrin recycling could be widely applicable and effective against many cancers. We also discuss possible therapeutic contexts where integrin trafficking can be effectively targeted, and what molecular interfaces may hopefully be druggable. (C) 2013 Elsevier Inc. All rights reserved.
• Co-Overexpression of GEP100 and AMAP1 Proteins Correlates with Rapid Local Recurrence after Breast Conservative Therapy

  Rumiko Kinoshita, Jin-Min Nam, Yoichi M. Ito, Kanako C. Hatanaka, Ari Hashimoto, Haruka Handa, Yutaro Otsuka, Shigeru Hashimoto, Yasuhito Onodera, Mitsuchika Hosoda, Shunsuke Onodera, Shinichi Shimizu, Shinya Tanaka, Hiroki Shirato, Mishie Tanino, Hisataka Sabe

  PLOS ONE 8 (10) e76791  1932-6203 2013/10 [Refereed][Not invited]
   

  A major problem of current cancer research and therapy is prediction of tumor recurrence after initial treatment, rather than the simple biological characterization of the malignancy and proliferative properties of tumors. Breast conservation therapy (BCT) is a well-approved, standard treatment for patients with early stages of breast cancer, which consists of lumpectomy and whole-breast irradiation. In spite of extensive studies, only 'age' and 'Ki-67 positivity' have been identified to be well correlated with local recurrence after BCT. An Arf6 pathway, activated by GEP100 under receptor tyrosine kinases (RTKs) and employs AMAP1 as its effector, is crucial for invasion and metastasis of some breast cancer cells. This pathway activates beta 1 integrins and perturbs E-cadherin-based adhesions, hence appears to be integral for epithelial-mesenchymal transdifferentiation (EMT). We here show that expression of the Arf6 pathway components statistically correlates with rapid local recurrence after BCT. We retrospectively analyzed four hundred seventy-nine patients who received BCT in Hokkaido University Hospital, and found 20 patients had local recurrence. We then analyzed pathological samples of patients who experienced local recurrence by use of Kaplan-Meier analysis, Stepwise regression analysis and the t-test, coupled with immunostaining, and found that co-overexpression of GEP100 and AMAP1 correlates with rapidity of the local recurrence. Their margin-status, node-positivity, and estrogen receptor (ER)-or progesterone receptor (PgR)positivity did not correlated with the rapidity. This study is the first to show that expression of a certain set of proteins correlates with the rapidity of local recurrence. Our results are useful not only for prediction, but highlight the possibility of developing novel strategies to block local recurrence. We also discuss why mRNAs encoding these proteins have not been identified to correlate with local recurrence by previous conventional gene expression profiling analyses.
• Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis

  T. Osawa, N. Ohga, K. Akiyama, Y. Hida, K. Kitayama, T. Kawamoto, K. Yamamoto, N. Maishi, M. Kondoh, Y. Onodera, M. Fujie, N. Shinohara, K. Nonomura, M. Shindoh, K. Hida

  BRITISH JOURNAL OF CANCER 109 (8) 2237 - 2247 0007-0920 2013/10 [Refereed][Not invited]
   

  Background: Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs. Methods: TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using beta-aminopropionitrile (BAPN). Results: LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model. Conclusion: LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.
• β1-integrin via NF-κB signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

  Jin-Min Nam, Kazi M. Ahmed, Sylvain Costes, Hui Zhang, Yasuhito Onodera, Adam B. Olshen, Kanako C. Hatanaka, Rumiko Kinoshita, Masayori Ishikawa, Hisataka Sabe, Hiroki Shirato, Catherine C. Park

  Breast Cancer Research 15 (4) R60  1465-5411 2013/07/25 [Refereed][Not invited]
   

  Introduction: Ductal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells.Methods: We measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence.Results: P-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, α5β1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-κB) to the β1-integrin promoter region, associated with up-regulation of α5β1-integrins. Inhibition of NF-κB or β1-integrin signaling abrogated emergence of the invasive activity.Conclusions: P-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by β1-integrin and NF-κB signaling. © 2013 Nam et al. licensee BioMed Central Ltd.
• beta 1-integrin via NF-kappa B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer

  Jin-Min Nam, Kazi M. Ahmed, Sylvain Costes, Hui Zhang, Yasuhito Onodera, Adam B. Olshen, Kanako C. Hatanaka, Rumiko Kinoshita, Masayori Ishikawa, Hisataka Sabe, Hiroki Shirato, Catherine C. Park

  BREAST CANCER RESEARCH 15 (4) 1465-542X 2013 [Refereed][Not invited]
   

  Introduction: Ductal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS and identified possible molecular mechanisms underlying invasive progression in surviving cells. Methods: We measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt) which, in three-dimensional laminin-rich extracellular matrix (lrECM) and in vivo, form organotypic DCIS-like lesions with lumina expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in three-dimensional lrECM, a malignant phenotype emerged creating a model for invasive recurrence. Results: P-Akt was up-regulated in clinical DCIS specimens and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in three-dimensional lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in three-dimensional lrECM, a malignant phenotype emerged, characterized by invasive activity, up-regulation of fibronectin, alpha 5 beta 1-integrin, matrix metalloproteinase-9 (MMP-9) and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of nuclear factor-kappa B (NF-kappa B) to the beta 1-integrin promoter region, associated with up-regulation of alpha 5 beta 1-integrins. Inhibition of NF-kappa B or beta 1-integrin signaling abrogated emergence of the invasive activity. Conclusions: P-Akt is up-regulated in some human DCIS lesions and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in three-dimensional lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in three-dimensional lrECM mediated by beta 1-integrin and NF-kappa B signaling.
• ['Molecular biology' of breast cancer].

  Onodera Y

  Nihon rinsho. Japanese journal of clinical medicine 70 Suppl 7 61 - 65 0047-1852 2012/09 [Refereed][Not invited]
  
• Rab5c promotes AMAP1-PRKD2 complex formation to enhance beta 1 integrin recycling in EGF-induced cancer invasion

  Yasuhito Onodera, Jin-Min Nam, Ari Hashimoto, Jim C. Norman, Hiroki Shirato, Shigeru Hashimoto, Hisataka Sabe

  JOURNAL OF CELL BIOLOGY 7 197 (7) 983 - 996 0021-9525 2012/06 [Refereed][Not invited]
   

  Epidermal growth factor receptor (EGFR) signaling is one of the crucial factors in breast cancer malignancy. Breast cancer cells often overexpress Arf6 and its effector, AMAP1/ASAP1/DDEF1; in these cells, EGFR signaling may activate the Arf6 pathway to induce invasion and metastasis. Active recycling of some integrins is crucial for invasion and metastasis. Here, we show that the Arf6-AMAP1 pathway links to the machinery that recycles beta 1 integrins, such as alpha 3 beta 1, to promote cell invasion upon EGFR stimulation. We found that AMAP1 had the ability to bind directly to PRKD2 and hence to make a complex with the cytoplasmic tail of the beta 1 subunit. Moreover, GTP-Rab5c also bound to AMAP1, and activation of Rab5c by EGFR signaling was necessary to promote the intracellular association of AMAP1 and PRKD2. Our results suggest a novel mechanism by which EGFR signaling promotes the invasiveness of some breast cancer cells via integrin recycling.
• Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells

  Yamamoto K, Ohga N, Hida Y, Maishi N, Kawamoto T, Kitayama K, Akiyama K, Osawa T, Kondoh M, Matsuda K, Onodera Y, Fujie M, Kaga K, Hirano S, Shinohara N, Shindoh M, Hida K

  Br J Cancer 106 (6) 1214 - 1223 0007-0920 2012 [Refereed][Not invited]
  
• Breast Cancer Cells in Three-dimensional Culture Display an Enhanced Radioresponse after Coordinate Targeting of Integrin alpha 5 beta 1 and Fibronectin

  Jin-Min Nam, Yasuhito Onodera, Mina J. Bissell, Catherine C. Park

  CANCER RESEARCH 70 (13) 5238 - 5248 0008-5472 2010/07 [Refereed][Not invited]
   

  Tactics to selectively enhance cancer radioresponse are of great interest. Cancer cells actively elaborate and remodel their extracellular matrix (ECM) to aid in survival and progression. Previous work has shown that beta 1-integrin inhibitory antibodies can enhance the growth-inhibitory and apoptotic responses of human breast cancer cell lines to ionizing radiation, either when cells are cultured in three-dimensional laminin-rich ECM (3D IrECM) or grown as xenografts in mice. Here, we show that a specific alpha heterodimer of beta 1-integrin preferentially mediates a prosurvival signal in human breast cancer cells that can be specifically targeted for therapy. 3D IrECM culture conditions were used to compare alpha-integrin heterodimer expression in malignant and nonmalignant cell lines. Under these conditions, we found that expression of alpha 5 beta 1-integrin was upregulated in malignant cells compared with nonmalignant breast cells. Similarly, we found that normal and oncofetal splice variants of fibronectin, the primary ECM ligand of alpha 5 beta 1-integrin, were also strikingly upregulated in malignant cell lines compared with nonmalignant acini. Cell treatment with a peptide that disrupts the interactions of alpha 5 beta 1-integrin with fibronectin promoted apoptosis in malignant cells and further heightened the apoptotic effects of radiation. In support of these results, an analysis of gene expression array data from breast cancer patients revealed an association of high levels of alpha 5-integrin expression with decreased survival. Our findings offer preclinical validation of fibronectin and alpha 5 beta 1-integrin as targets for breast cancer therapy. Cancer Res; 70(13); 5238-48. (C) 2010 AACR.
• The EGFR-GEP100-Arf6-AMAP1 Signaling Pathway Specific to Breast Cancer Invasion and Metastasis(dagger)

  Hisataka Sabe, Shigeru Hashimoto, Masaki Morishige, Eiji Ogawa, Ari Hashimoto, Jin-Min Nam, Koichi Miura, Hajime Yano, Yasuhito Onodera

  TRAFFIC 10 (8) 982 - 993 1398-9219 2009/08 [Refereed][Not invited]
   

  Tumors are tissue-specific diseases, and their mechanisms of invasion and metastasis are highly diverse. In breast cancer, biomarkers that specifically correlate with the invasive phenotypes have not been clearly identified. A small GTPase Arf6 primarily regulates recycling of plasma membrane components. We have shown that Arf6 and its effector AMAP1 (DDEF1, DEF1, ASAP1 and centaurin beta 4) are abnormally overexpressed in some breast cancers and used for their invasion and metastasis. Overexpression of these proteins is independent of the transcriptional upregulation of their genes, and occurs only in highly malignant breast cancer cells. We recently identified GEP100 (BRAG2) to be responsible for the Arf6 activation to induce invasion and metastasis, by directly binding to ligand-activated epidermal growth factor receptor (EGFR). A series of our studies revealed that for activation of the invasion pathway of EGFR, it is prerequisite that Arf6 and AMAP1 both are highly overexpressed, and that EGFR is activated by ligands. Pathological analyses indicate that a significant large population of human ductal cancers may utilize the EGFR-GEP100-Arf6-AMAP1 pathway for their malignancy. Microenvironments have been highly implicated in the malignancy of mammary tumors. Our results reveal an aspect of the precise molecular mechanisms of some breast cancers, in which full invasiveness is not acquired just by intracellular alterations of cancer cells, but extracellular factors from microenvironments may also be necessary. Possible translation of our knowledge to cancer therapeutics will also be discussed.
• 【シグナル伝達研究 2008'09 疾患発症の分子メカニズムと実現化する分子標的薬開発】シグナル伝達研究 因子から現象へ がんの浸潤形質獲得過程における低分子量Gタンパク質Arf6シグナル伝達

  橋本 茂, 森重 真毅, 小川 栄治, 橋本 あり, 小野寺 康仁, 佐邊 壽孝

  実験医学 (株)羊土社 26 (15) 2349 - 2355 0288-5514 2008/09 

  がんの主たる脅威は、浸潤・転移性の獲得にある。恒常性を維持した細胞浸潤は、胎児期の発生・分化過程や創傷治癒、免疫応答などでみられるが、どのような制御機構の破綻ががんの浸潤形質を誘導しているのか未だ不明のままである。がんの浸潤形質獲得は、がん細胞自身のgenetic/epigeneticな変異の多段階的蓄積のみならず、微小環境の変化に依存している。われわれは、低分子量Gタンパク質Arf6を中心としたシグナル伝達経路の活性化が浸潤形質の獲得に必須であることを見出した。本稿では、がん浸潤形質の誘導に関連した最近の知見を概説するとともに、われわれのモデルについて紹介したい。(著者抄録)
• Fbx8 makes Arf6 refractory to function via ubiquitination

  Hajime Yano, Itaru Kobayashi, Yasuhito Onodera, Frederic Luton, Michel Franco, Yuichi Mazaki, Shigeru Hashimoto, Kazuhiro Iwai, Ze'ev Ronai, Hisataka Sabe

  MOLECULAR BIOLOGY OF THE CELL 19 (3) 822 - 832 1059-1524 2008/03 [Refereed][Not invited]
   

  The small GTP-binding protein Arf6 regulates membrane remodeling at cell peripheries and plays crucial roles in higher orders of cellular functions including tumor invasion. Here we show that Fbx8, an F-box protein bearing the Sec7 domain, mediates ubiquitination of Arf6. This ubiquitination did not appear to be linked to immediate proteasomal degradation of Arf6, whereas Fbx8 knockdown caused hyperactivation of Arf6. Expression of Fbx8 protein was substantially lost in several breast tumor cell lines, in which Arf6 activity is pivotal for their invasion. Forced expression of Fbx8 in these cells suppressed their Arf6 activities and invasive activities, in which the F-box and Sec7 domains of Fbx8 are required. Together with the possible mechanism as to how Fbx8-mediated ubiquitination interferes with the functions of Arf6, we propose that Fbx8 provides a novel suppressive control of Arf6 activity through noncanonical ubiquitination. Our results indicate that dysfunction of Fbx8 expression may contribute to the invasiveness of some breast cancer cells.
• [Breast neoplasms: invasion and metastasis].

  Onodera Y, Nam JM

  Nihon rinsho. Japanese journal of clinical medicine 65 Suppl 6 33 - 38 0047-1852 2007/06 [Refereed][Not invited]
  
• CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery

  Jin-Min Nam, Yasuhito Onodera, Yuichi Mazaki, Hiroyuki Miyoshi, Shigeru Hashimoto, Hisataka Sabe

  EMBO JOURNAL 26 (3) 647 - 656 0261-4189 2007/02 [Refereed][Not invited]
   

  Expression of AMAP1 correlates well with the invasive phenotypes and malignancy of human primary breast carcinomas. AMAP1 recruits its binding proteins, such as cortactin and paxillin, to sites of Arf6 activation to form invadopodia. A mouse ortholog of AMAP1, ASAP1, is known to bind to CIN85, a binding partner of an E3 ligase, Cbl. Here, we found that CIN85 colocalizes with AMAP1 at invadopodia, and binding of AMAP1 with CIN85 is important for the invasive activities of breast cancer cells, including MDA-MB-231. siRNA-mediated silencing of CIN85, as well as Cbl, also inhibited the invasion. We moreover found that AMAP1 is monoubiquitinated, rather than polyubiquitinated, by virtue of Cbl and provide evidence that the ability of AMAP1 to be monoubiquitinated is important for its involvement in invasion. Our results indicate that CIN85, as well as Cbl, which is a well-known suppressor of growth factor receptor signaling, can be positively involved in tumor invasion, and suggest that a complex epigenetic process is involved in AMAP1 function in breast cancer cell invasion.
• ArfGAP family proteins in cell adhesion, migration and tumor invasion

  Hisataka Sabe, Yasuhito Onodera, Yuichi Mazaki, Shigeru Hashimoto

  CURRENT OPINION IN CELL BIOLOGY 18 (5) 558 - 564 0955-0674 2006/10 [Refereed][Not invited]
   

  The identification of several ArfGAP proteins as binding partners of paxillin, an integrin signaling and scaffolding protein, has suggested the existence of molecular links between integrin functions and intracellular traffic, as proposed by MS Bretscher long ago. Among the paxillin-binding ArfGAPs, AMAP1 has recently been strongly implicated in tumor invasion as well as malignancy, owing to its highly augmented expression in tumors and its direct involvement in invasive activities. Another ArfGAP, Git2, was found to be a component of the G beta gamma-mediated directional sensing machinery, while simultaneously playing an essential role in the suppressive control of superoxide production, which is mediated by vesicle transport in GPCR-stimulated neutrophils. These emerging molecular mechanisms may further delineate key processes regulating intracellular traffic as principal controls of cell motility and invasive activities.
• [Breast cancer invasiveness: how does it emerge?].

  Hashimoto S, Onodera Y

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 6 Suppl 51 803 - 810 0039-9450 2006/05 [Refereed][Not invited]
  
• Regulation of epithelial cell-cell interaction by Arf6 signaling

  Miura Koichi, Yano Hajime, Onodera Yasuhito, Kojima Chie, Hashimoto Shigeru, Sabe Hisataka

  CELL STRUCTURE AND FUNCTION 30 5  0386-7196 2005/06 [Refereed][Not invited]
  
• Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities

  Y Onodera, S Hashimoto, A Hashimoto, M Morishige, Y Mazaki, A Yamada, E Ogawa, M Adachi, T Sakurai, T Manabe, H Wada, N Matsuura, H Sabe

  EMBO JOURNAL 24 (5) 963 - 973 0261-4189 2005/03 [Refereed][Not invited]
   

  Identification of the molecular machinery employed in cancer invasion, but not in normal adult cells, will greatly contribute to cancer therapeutics. Here we found that an ArfGAP, AMAP1/PAG2, is expressed at high levels in highly invasive breast cancer cells, but at very low levels in noninvasive breast cancer cells and normal mammary epithelial cells. siRNA-mediated silencing of AMAP1 effectively blocked the invasive activities. AMAP1 expression in human breast primary tumors also indicated its potential correlation with malignancy. Paxillin and cortactin have been shown to colocalize at invadopodia and play a pivotal role in breast cancer invasion. We found that AMAP1 is also localized at invadopodia, and acts to bridge paxillin and cortactin. This AMAP1-mediated trimeric protein complex was detected only in invasive cancer cells, and blocking this complex formation effectively inhibited their invasive activities in vitro and metastasis in mice. Our results indicate that AMAP1 is a component involved in invasive activities of different breast cancers, and provide new information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis.
• Assays and properties of the ArfGAPs, AMAP1 and AMAP2, in Arf6 function

  S Hashimoto, A Hashimoto, A Yamada, Y Onodera, H Sabe

  GTPASES REGULATING MEMBRANE DYNAMICS 404 216 - 231 0076-6879 2005 [Refereed][Not invited]
   

  The GTPase-activating protein (GAP) domain for Arfs primarily consists of a zinc-finger structure, which is not present in known GAPS for the other Ras-superfamily GTPases. More than 20 genes have been found to encode proteins bearing the ArfGAP domain in the human genome: a number that is much larger than that of the Arf isoforms. Several Arf isoforms, such as Arf1 and Arf6, indeed have been shown to each employ multiple different ArfGAPs for their regulation and function. We have found that two ArfGAPs, namely AMAP1 and AMAP2, exhibit a novel biochemical property of directly and selectively binding to GTP-Arf6 without immediate GAPing activity, while they were previously shown to exhibit efficient catalytic GAPing activities to Arf isoforms except Arf6 in vitro. Such property of AMAPs appears to be important for AMAPs-mediated recruitment of auxiliary molecules, including paxillin, cortactin, amphiphysin, and intersectin, to sites of Arf6 activation. AMAPs thus appear to act as "effectors" rather than simple GAPS in some aspects of Arf6 function. This article presents methods and protocols developed for the functional characterization of AMAPs in Arf6 function. These methods may be applied to other types of ArfGAPs to further clarify the cellular functions of ArfGAPs as well as Arfs.
• Requirement for Arf6 in breast cancer invasive activities

  S Hashimoto, Y Onodera, A Hashimoto, M Tanaka, M Hamaguchi, A Yamada, H Sabe

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (17) 6647 - 6652 0027-8424 2004/04 [Refereed][Not invited]
   

  in most human breast cancer cell lines, there is a direct correlation between their in vivo invasive phenotypes and in vitro invasion activities. Here, we found that ADP-ribosylation factor 6 (Arf6) is localized at the invadopodia of the cultured breast cancer cells MDA-MB-231, and its suppression by a small-interfering RNA duplex effectively blocks the invasive activities of the cells, such as invadopodia formation, localized matrix degradation and Matrigel transmigration but not the cell-adhesion activity. We also found that the GTP hydrolysis-defective mutant Arf6(Q67L) and the GTP-binding defective mutant Arf6(T27N) both blocked these invasive activities but not cell adhesion, suggesting the necessity of continued activation and cycling of the Arf6 GTPase cycle in invasion. Among the different human breast cancer cell lines that we examined, cell lines with high invasive activities expressed higher amounts of Arf6 protein than those in weakly invasive and noninvasive cell lines, although no notable correlation was found between Arf6 mRNA expression levels and invasive activities. Moreover, Matrigel-transmigration activity of all of these invasive cells was blocked effectively by an Arf6 small-interfering RNA duplex. Hence, Arf6 appears to be an integral component of breast cancer invasive activities, and we propose that Arf6 and the intracellular machinery regulating Arf6 during invasion should be considered as therapeutic targets for the prevention of breast cancer invasion.
• Interaction of POB1, a downstream molecule of small G protein Ral, with PAG2, a paxillin-binding protein, is involved in cell migration

  T Oshiro, S Koyama, S Sugiyama, A Kondo, Y Onodera, T Asahara, H Sabe, A Kikuchi

  JOURNAL OF BIOLOGICAL CHEMISTRY 277 (41) 38618 - 38626 0021-9258 2002/10 [Refereed][Not invited]
   

  POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl-terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl-terminal region including the SH3 domain of PAG2. Substitutions of Pro(423) and Pro(426) with Ala (POB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.

MISC

• Macroscopic order formation in myoblast populations driven by intracellular molecular dynamics

  麓佳月, 高田真吾, 小野寺康仁, 川口喬吾, 畠山鎮次, 佐邊壽孝, 及川司  日本分子生物学会年会プログラム・要旨集(Web)  45th-  2022
• 好中球様細胞に分化させたHL-60細胞のケモタキシスにおける小胞体とミトコンドリアの接触

  真崎雄一, 高田真吾, 小林純子, 前川聡, 小野寺康仁, 佐邊壽孝  日本分子生物学会年会プログラム・要旨集(Web)  43rd-  2020
• p53 counteracts EZH2 at the nuclear lamina to prevent H3K27 hypermethylation

  及川司, 大西なおみ, 小野寺康仁, 橋本あり, 植田幸嗣, 佐邊壽孝  日本生化学会大会(Web)  93rd-  2020
• MFN2は好中球様細胞に分化させたHL-60細胞のケモタキシスに関与する

  真崎雄一, 高田真吾, 小林純子, 前川聡, 東恒仁, 小野寺康仁, 佐邊壽孝  日本分子生物学会年会プログラム・要旨集(Web)  42nd-  2019
• Requirement for p53 in intra-nuclear dynamics of the K27-trimethylated histone H3 during DNA replication

  及川司, 大西なおみ, 小野寺康仁, 橋本あり, 植田幸嗣, 佐邊壽孝  日本生化学会大会(Web)  92nd-  2019
• 膵癌ドライバー変異はARF6-AMAP1経路を活性化し悪性度と免疫回避能を促進する(Pancreatic KRAS and TP53 oncogenes cooperatively activate ARF6-AMAP1 pathway to drive malignancy and immune evasion)

  橋本 あり, 橋本 茂, 古川 聖太郎, 蔦保 暁生, 小野寺 康仁, 大塚 勇太郎, 半田 悠, 及川 司, 水上 裕輔, 村上 正晃, 平野 聡, 佐邊 壽孝  日本癌学会総会記事  77回-  2219  -2219  2018/09  [Not refereed][Not invited]
  
• エンドセリンB受容体はGRP78と相互作用する

  真崎雄一, 東恒仁, 堀之内孝広, 橋本あり, 橋本茂, 南ジンミン, 小野寺康仁  日本分子生物学会年会プログラム・要旨集(Web)  41st-  2018
• 上皮細胞においてp53はE-cadherin遺伝子発現制御部位に結合し、EZH2による発現抑制に拮抗する

  及川 司, 大塚 勇太郎, 小野寺 康仁, 堀川 芽衣, 橋本 あり, 橋本 茂, 鈴木 穣, 佐邊 壽孝  生命科学系学会合同年次大会  2017年度-  [3PT18  -06(3P  2017/12  [Not refereed][Not invited]
  
• 膵癌ドライバー変異はARF6経路を介して癌悪性度とPD‐L1発現を促進する

  橋本あり, 橋本茂, 古川聖太郎, 古川聖太郎, 蔦保暁生, 蔦保暁生, 大塚勇太郎, 半田悠, 小野寺康仁, 及川司, 平野聡, 佐邊壽孝  日本生化学会大会(Web)  90th-  ROMBUNNO.1P‐1028 (WEB ONLY)  -1028]  2017/12  [Not refereed][Not invited]
  
• がんの浸潤・転移研究の新機軸 Arf6経路 難治性癌の悪性度進展・抗癌剤抵抗性に根幹的経路

  佐邊 壽孝, 橋本 あり, 小野寺 康仁, 及川 司, 橋本 茂  日本癌学会総会記事  76回-  S1  -2  2017/09  [Not refereed][Not invited]
  
• 上皮形質安定性をp53に依存する上皮細胞と依存しない上皮細胞の差異に関する解析

  及川 司, 大塚 勇太郎, 小野寺 康仁, 堀川 芽衣, 橋本 あり, 橋本 茂, 鈴木 穣, 佐邊 壽孝  日本癌学会総会記事  76回-  P  -1107  2017/09  [Not refereed][Not invited]
  
• Targeting integrins with RGD-conjugated gold nanoparticles in radiotherapy decreases the invasive activity of breast cancer cells

  Ping-Hsiu Wu, Yasuhito Onodera, Yuki Ichikawa, Erinn B. Rankin, Amato J. Giaccia, Yuko Watanabe, Wei Qian, Takayuki Hashimoto, Hiroki Shirato, Jin-Min Nam  International Journal of Nanomedicine  12-  5069  -5085  2017/07/14  [Not refereed][Not invited]
   

  Gold nanoparticles (AuNPs) have recently attracted attention as clinical agents for enhancing the effect of radiotherapy in various cancers. Although radiotherapy is a standard treatment for cancers, invasive recurrence and metastasis are significant clinical problems. Several studies have suggested that radiation promotes the invasion of cancer cells by activating molecular mechanisms involving integrin and fibronectin (FN). In this study, polyethylene-glycolylated AuNPs (P-AuNPs) were conjugated with Arg-Gly-Asp (RGD) peptides (RGD/P-AuNPs) to target cancer cells expressing RGD-binding integrins such as α5-and αv-integrins. RGD/P-AuNPs were internalized more efficiently and colocalized with integrins in the late endosomes and lysosomes of MDA-MB-231 cells. A combination of RGD/P-AuNPs and radiation reduced cancer cell viability and increased DNA damage compared to radiation alone in MDA-MB-231 cells. Moreover, the invasive activity of breast cancer cell lines after radiation treatment was significantly inhibited in the presence of RGD/P-AuNPs. Microarray analyses revealed that the expression of FN in irradiated cells was suppressed by combined use of RGD/P-AuNPs. Reduction of FN and downstream signaling may be involved in suppressing radiation-induced invasive activity by RGD/P-AuNPs. Our study suggests that RGD/P-AuNPs can target integrin-overexpressing cancer cells to improve radiation therapy by suppressing invasive activity in addition to sensitization. Thus, these findings provide a possible clinical strategy for using AuNPs to treat invasive breast cancer following radiotherapy.
• がん細胞のミトコンドリア分布制御による浸潤亢進と酸化ストレス回避

  小野寺康仁, 佐邊壽孝  臨床ストレス応答学会大会抄録集  12th-  2017
• 好中球のケモタキシスにおいて,ARF1の活性化は,ARF1‐RAC1の相互制御回路を開始する

  真崎雄一, 小野寺康仁, 東恒仁, 堀之内孝広, 及川司, 佐邊壽孝  日本細胞生物学会大会(Web)  69th-  ROMBUNNO.T8‐11(P1‐077) (WEB ONLY)  -63  2017  [Not refereed][Not invited]
  
• Gold Nanoparticles With RGD Peptide in Radiation Therapy Suppress the Invasion Activity of Breast Cancer Cells

  P. H. Wu, Y. Onodera, Y. Ichikawa, Y. Watanabe, W. Qian, T. Hashimoto, H. Shirato, J. M. Nam  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  96-  (2)  E574  -E574  2016/10  [Not refereed][Not invited]
  
• p53はEZH2と機能的に競合することで上皮性維持に寄与する

  及川 司, 大塚 勇太郎, 小野寺 康仁, 半田 悠, 橋本 あり, 橋本 茂, 鈴木 穣, 佐邊 壽孝  日本癌学会総会記事  75回-  J  -3030  2016/10  [Not refereed][Not invited]
  
• メバロン酸経路阻害剤スタチンはArf6経路を高発現する癌に有効である

  橋本 あり, 橋本 茂, 及川 司, 大塚 勇太郎, 半田 悠, 小野寺 康仁, 佐邊 壽孝  日本生化学会大会プログラム・講演要旨集  89回-  [1P  -268]  2016/09  [Not refereed][Not invited]
  
• がん形質の獲得と代謝の局所制御

  小野寺康仁, 堀川芽衣, 佐邊壽孝  がんと代謝研究会プログラム&抄録集  4th-  44  2016/07  [Not refereed][Not invited]
  
• 代謝と細胞動態 細胞動態・運命決定を司る内なるチカラ 代謝と輸送および区画化を介したがん形質の誘導

  小野寺 康仁, Bissell Mina, 佐邊 壽孝  日本細胞生物学会大会講演要旨集  68回-  21  -21  2016/05  [Not refereed][Not invited]
  
• 膵癌細胞の浸潤・転移および化学療法抵抗性メカニズムの解明

  古川聖太郎, 橋本あり, 橋本茂, 小野寺康仁, 及川司, 大塚勇太郎, 佐邊壽孝, 平野聡  日本外科学会定期学術集会(Web)  116th-  PS-002-2 (WEB ONLY)  -002  2016/04  [Not refereed][Not invited]
  
• がん細胞における小胞輸送経路を介したミトコンドリア分布制御と活性酸素および放射線への耐性

  小野寺康仁, 南ジンミン, 白土博樹, 佐邊壽孝  日本放射線影響学会大会抄録(Web)  59th-  ROMBUNNO.W12‐1 (WEB ONLY)  -26  2016  [Not refereed][Not invited]
  
• p53はエピジェネティック制御を介して上皮性を維持する

  及川 司, 小野寺 康仁, 大塚 勇太郎, 半田 悠, 橋本 あり, 橋本 茂, 鈴木 穣, 佐邊 壽孝  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集  88回・38回-  [2P1108]  -[2P1108]  2015/12  [Not refereed][Not invited]
  
• Arf6-AMAP1経路によるROS制御は乳癌の放射線抵抗性に寄与する

  小野寺 康仁, 南 ジンミン, 及川 司, 白土 博樹, 佐邊 壽孝  日本癌学会総会記事  74回-  E  -1049  2015/10  [Not refereed][Not invited]
  
• 放射線照射後の乳腺上皮細胞の3次元構造維持に関わる分子機序の解析

  南 ジンミン, 小野寺 康仁, 佐邊 壽孝, 白土 博樹  日本癌学会総会記事  74回-  P  -2343  2015/10  [Not refereed][Not invited]
  
• Arf6-AMAP1経路によるROS制御は乳癌の放射線抵抗性に寄与する

  小野寺 康仁, 南 ジンミン, 及川 司, 白土 博樹, 佐邊 壽孝  日本癌学会総会記事  74回-  E  -1049  2015/10  [Not refereed][Not invited]
  
• 放射線照射後の乳腺上皮細胞の3次元構造維持に関わる分子機序の解析

  南 ジンミン, 小野寺 康仁, 佐邊 壽孝, 白土 博樹  日本癌学会総会記事  74回-  P  -2343  2015/10  [Not refereed][Not invited]
  
• The invasion activity after radiation treatment is suppressed by RGD peptide conjugated gold nanoparticles in breast cancer cells

  P. Wu, Y. Onodera, Y. Ichikawa, Y. Watanabe, W. Qian, T. Hashimoto, H. Shirato, J. Nam  MOLECULAR BIOLOGY OF THE CELL  26-  2015  [Not refereed][Not invited]
  
• 糖代謝と小胞輸送の相互作用を介したがん形質の誘導

  小野寺康仁, 南ジンミン, 白土博樹, BISSELL Mina, 佐邊壽孝  がんと代謝研究会プログラム&抄録集  3rd-  44  2015  [Not refereed][Not invited]
  
• Arf6-AMAP1経路による酸化還元状態の恒常性維持は乳癌の放射線抵抗性に寄与する(Robust redox homeostasis mediated by Arf6-AMAP1 pathway confers resistance to ionizing radiation in breast cancer)

  小野寺 康仁, 南 ジンミン, 及川 司, 白土 博樹, 佐邊 壽孝  日本癌学会総会記事  73回-  E  -3010  2014/09  [Not refereed][Not invited]
  
• 乳癌において変異p53がリガンド反応性の間葉型浸潤分子装置を創出する機序(TP53 alterations generate Arf6-based mesenchymal invasion pathway that is activated by RTKs and TGFβ1 in breast cancer)

  橋本 あり, 橋本 茂, 杉野 弘和, 吉河 歩, 及川 司, 小野寺 康仁, 半田 悠, 大塚 勇太郎, 岩見 昴亮, 小根山 千歳, 岡田 雅人, 福田 光則, 佐邊 壽孝  日本癌学会総会記事  73回-  J  -2077  2014/09  [Not refereed][Not invited]
  
• p53は間葉系形質を持つ乳がん細胞に上皮系形質を再獲得させる(p53 recalls epithelial memory in mammary cancer cells with mesenchymal phenotypes)

  及川 司, 小野寺 康仁, 橋本 あり, 橋本 茂, 佐邊 壽孝  日本癌学会総会記事  73回-  P  -1159  2014/09  [Not refereed][Not invited]
  
• 乳癌における放射線照射後の浸潤能獲得過程に関わる分子機序の解析(Analysis of molecular mechanism involved in invasiveness of radiation treated breast cancer cells)

  南 ジンミン, 小野寺 康仁, 佐邊 壽孝, 白土 博樹  日本癌学会総会記事  73回-  P  -1450  2014/09  [Not refereed][Not invited]
  
• 癌放射線治療への分子生物学的アプローチ 変異p53が放射線抵抗性に根幹的な間葉型浸潤経路を創出する機構(Toward the improvement of radiotherapy: Approaches from the molecular biological point of view Mechanisms by which oncogenic mutant-p53 generates mesenchymal invasive pathway pivotal to a radiation resis

  佐邊 壽孝, 橋本 あり, 橋本 茂, 小野寺 康仁, 及川 司, Nam Jin-Min, 小根山 千歳, 杉野 弘和, 吉河 歩, 大塚 勇太郎, 半田 悠, 芳野 正修, 岡田 雅人  日本癌学会総会記事  72回-  64  -64  2013/10  [Not refereed][Not invited]
  
• 放射線照射後の乳癌再発に関わるシグナルの解析(Possible mechanisms of non-invasive to invasive phenotypic conversion of breast cancer cells upon radiation)

  南 ジンミン, 小野寺 康仁, 石川 正純, 佐邊 壽孝, 白土 博樹  日本癌学会総会記事  72回-  219  -219  2013/10  [Not refereed][Not invited]
  
• 細胞が持つリサイクルシステム研究の新展開 p53変異によるGEP100-Arf6-AMAP1経路の活性化と乳癌の浸潤形質獲得

  橋本 茂, 橋本 あり, 小根山 千歳, 吉河 歩, 杉野 弘和, 半田 悠, 芳野 正修, 大塚 勇太郎, 小野寺 康仁, 岡田 雅人, 佐邊 壽孝  日本生化学会大会プログラム・講演要旨集  86回-  2S04a  -3  2013/09  [Not refereed][Not invited]
  
• 乳癌の分子生物学と発癌機序 分子生物学 乳癌の‘分子生物学’:概論

  小野寺康仁  日本臨床  70-  61-65  2012/09/20  [Not refereed][Not invited]
  
• 乳癌浸潤に中枢的なArf6経路は変異p53により創出される(Mutant-p53 generates GEP100-Arf6-AMAP1 pathway to promote breast cancer cell invasiveness in response to TGFbeta1)

  橋本 あり, 橋本 茂, 吉河 歩, 杉野 弘和, 半田 悠, 味藤 静, 佐藤 宏紀, 大塚 勇太郎, 芳野 日南子, 南 ジンミン, 小野寺 康仁, 佐邊 壽孝  日本癌学会総会記事  71回-  399  -399  2012/08  [Not refereed][Not invited]
  
• 癌浸潤におけるAMAP1-PRKD2複合体によるインテグリンリサイクリングとその制御機構(beta1 integrin recycling via AMAP1-PRKD2 complex regulated by small GTPases in cancer invasion)

  小野寺 康仁, 南 ジンミン, 橋本 あり, 白土 博樹, 橋本 茂, 佐邊 壽孝  日本癌学会総会記事  71回-  421  -421  2012/08  [Not refereed][Not invited]
  
• 変異p53が癌浸潤転移シグナル経路を創出し活性化する

  橋本あり, 橋本茂, 吉河歩, 杉野弘和, 半田悠, 木下留美子, 畑中佳奈子, 三上修治, 谷野美智枝, 味藤静, 佐藤宏紀, 大塚勇太郎, 芳野日南子, 加戸由加里, NAM Jin‐Min, 小野寺康仁, 田中伸哉, 白土博樹, 佐邊壽孝  日本分子生物学会年会プログラム・要旨集(Web)  35th-  2W10II-1 (WEB ONLY)  2012  [Not refereed][Not invited]
  
• EGF刺激による乳癌細胞浸潤におけるAMAP1の詳細な作用機構(AMAP1 promotes β1 integrin recycling via PRKD2 and Rab5c in EGF-induced invasion of breast cancer cells)

  小野寺 康仁, 南 ジンミン, 橋本 茂, 橋本 あり, 白土 博樹, 佐邊 壽孝  日本癌学会総会記事  70回-  37  -38  2011/09  [Not refereed][Not invited]
  
• 変異p53はArf6活性化経路を介した浸潤獲得形質に必須である(Mutant p53 is essential for TGFβ1-induced breast cancer cell invasiveness via activation of GEP100-Arf6-AMAP1 pathway)

  橋本 あり, 橋本 茂, 大塚 勇太郎, 吉河 歩, 杉野 弘和, 半田 悠, 南 ジンミン, 佐藤 宏紀, 福田 諭, 小野寺 康仁, 佐邊 壽孝  日本癌学会総会記事  70回-  38  -38  2011/09  [Not refereed][Not invited]
  
• TGFβ及び低酸素によるArf6活性化を介した癌浸潤形質獲得におけるエピジェネティック因子の関与(EZH2 is essential to Arf6 activation necessary for TGFβ1- and hypoxia-induced invasiveness of breast cancer cells)

  橋本 茂, 橋本 あり, 小野寺 康仁, 大塚 勇太郎, 吉河 歩, 杉野 弘和, 半田 悠, 佐藤 宏紀, 福田 諭, 毛受 暁史, 佐邊 壽孝  日本癌学会総会記事  70回-  77  -77  2011/09  [Not refereed][Not invited]
  
• 癌の悪性化における糖代謝と小胞輸送の役割(Glucose metabolism and intracellular trafficking in tumor malignancy)

  小野寺 康仁, 南 ジンミン, 橋本 茂, 橋本 あり, 佐邊 壽孝, Bissell Mina  日本細胞生物学会大会講演要旨集  63回-  119  -119  2011/05  [Not refereed][Not invited]
  
• TGFβ1による癌的EMTにおけるGEP100-Arf6-AMAP1シグナルの機能解析(HGFR/c-Met-mediated activation of GEP100-Arf6-AMAP1 pathway is an integral part for TGFβ-induced cancerous EMT and invasiveness)

  橋本 あり, 橋本 茂, 大塚 勇太郎, 佐藤 宏紀, 杉野 弘和, 吉河 歩, 梅本 勉, 小野寺 康仁, 福田 諭, 佐邊 壽孝  日本細胞生物学会大会講演要旨集  63回-  151  -151  2011/05  [Not refereed][Not invited]
  
• TGFβ1はGEP100-Arf6-AMAP1経路の活性化によりEMTを誘導し、この活性化は癌幹細胞性と関連する(TGFβ1 activates GEP100-Arf6-AMAP1 pathway to induce EMT, and possible relationship of this activation to cancer stemness)

  橋本 あり, 平野 真理子, 谷野 美智枝, 梅本 勉, 小野寺 康仁, 佐藤 宏紀, 木下 留美子, 南 ジンミン, 大塚 勇太郎, 福田 諭, 白土 博樹, 相沢 慎一, 橋本 茂, 田中 伸哉, 佐邊 壽孝  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  83回・33回-  2P  -0237  2010/12  [Not refereed][Not invited]
  
• 癌の悪性化における小胞輸送と糖代謝の役割(Intracellular trafficking and glucose metabolism in tumor malignancy)

  小野寺 康仁, 佐邊 壽孝  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  83回・33回-  3P  -1019  2010/12  [Not refereed][Not invited]
  
• 癌の悪性化における小胞輸送と糖代謝の役割(Roles of intracellular trafficking and glucose metabolism in tumor malignancy)

  小野寺 康仁, 佐邊 壽孝  日本癌学会総会記事  69回-  83  -83  2010/08  [Not refereed][Not invited]
  
• 上皮間葉転換 Arf6-AMAP1経路は癌的EMTに寄与する(EMT (Epithelial Mesenchymal Transition) The Arf6-AMAP1 pathway contributes to cancerous EMT)

  佐邊 壽孝, 小野寺 康仁, 橋本 あり, 橋本 茂  日本癌学会総会記事  69回-  240  -240  2010/08  [Not refereed][Not invited]
  
• 低酸素下の癌細胞の浸潤形質獲得とArf6活性化との関連(Hypoxia-induced invasive activity of breast cancer cells involves Arf6 activation)

  橋本 茂, 橋本 あり, 小野寺 康仁, 梅本 勉, 佐藤 宏紀, 毛受 暁史, 佐邊 壽孝  日本癌学会総会記事  69回-  418  -419  2010/08  [Not refereed][Not invited]
  
• TGFβによって誘導される癌的EMTにおけるGEP100-Arf6-AMAP1シグナルとHGFRとの相互作用(HGFR-mediated GEP100-Arf6-AMAP1 pathway is an integral part for TGFβ-induced cancerous EMT and invasiveness)

  橋本 あり, 平野 真理子, 梅本 勉, 小野寺 康仁, 佐藤 宏紀, 大塚 勇太郎, 橋本 茂, 佐邊 壽孝  日本癌学会総会記事  69回-  419  -419  2010/08  [Not refereed][Not invited]
  
• がん増殖を解くシグナル伝達研究の新展開 癌の悪性化における小胞輸送と糖代謝の役割(New frontiers of signal transduction toward understanding cancer growth Intracellular traffic and glucose metabolism in tumor malignancy)

  小野寺 康仁, 佐邊 壽孝  日本細胞生物学会大会講演要旨集  62回-  105  -105  2010/05  [Not refereed][Not invited]
  
• 低酸素下におけるArf6活性化と癌の浸潤,EMT進行,幹細胞化との関連性

  橋本茂, 橋本あり, 小野寺康仁, 梅本勉, 佐藤宏紀, 佐藤宏紀, 毛受暁史, 伊達洋至, 福田諭, 佐邊壽孝  生化学  83回・33回-  ROMBUNNO.3T4-10  -10  2010  [Not refereed][Not invited]
  
• シグナル伝達研究 I:因子から現象へ 4.がんの浸潤形質獲得過程における低分子量Gタンパク質Arf6シグナル伝達

  橋本茂, 森重真毅, 小川栄治, 橋本あり, 小野寺康仁, 佐邊壽孝  実験医学  26-  (15)  2349-2355  2008/09/15  [Not refereed][Not invited]
  
• GEP100‐Arf6‐AMAP1‐cortactinシグナル経路は癌浸潤と血管新生に共通である

  橋本あり, 橋本茂, 小川栄治, 小川栄治, 廣瀬まゆみ, 森重真毅, 毛受暁史, 小野寺康仁, 渋谷正史, 佐邊壽孝  生化学  81回・31回-  4S2-3  -3  2008  [Not refereed][Not invited]
  
• 乳癌浸潤転移におけるEGFR‐GEP100‐Arf6‐AMAP1経路:微小環境との相互作用

  佐邊壽孝, 橋本茂, 森重真毅, 橋本あり, 小川栄二, 矢野元, NAM Jinmin, 小野寺康仁  生化学  81回・31回-  4S14-3  -3  2008  [Not refereed][Not invited]
  
• Fbx8によるユビキチン化を介するArf6の抑制的制御と上皮組織形態形成との関連の可能性について(Fbx8 makes Arf6 refractory to function via ubiquitination: implication in epithelial tissue organization)

  矢野 元, 小野寺 康仁, 鳥井 郁子, 真崎 雄一, 橋本 茂, 辻村 亨, 佐邊 壽孝  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  80回・30回-  1T21  -3  2007/11  [Not refereed][Not invited]
  
• 基礎研究 III.病理・分子生理学 浸潤・転移

  小野寺康仁, NAM Jin‐Min  日本臨床  65-  33-38  2007/06/28  [Not refereed][Not invited]
  
• Invasion and metastasis

  小野寺 康仁, Nam Jin-Min  Japanese journal of clinical medicine  65-  (0)  33  -38  2007/06  [Not refereed][Not invited]
  
• CIN85, a Cbl-interacting multiadaptor protein, is a component of AMAP1-mediated breast cancer invasion machinery.

  J. Nam, Y. Onodera, Y. Mazaki, H. Miyoshi, S. Hashimoto, H. Sabe  Nat. Immunol.  7-  724  -731  2007  [Not refereed][Not invited]
  
• 乳癌細胞の浸潤におけるAMAP1のユビキチン化の役割(Property of AMAP1 to be monoubiquitinated via binding with CIN85 and Cbl is crucial for breast cancer invasive activity)

  Nam Jin-Min, 小野寺 康仁, 橋本 茂, 佐邊 壽孝  日本癌学会総会記事  65回-  295  -296  2006/09  [Not refereed][Not invited]
  
• 癌 5.癌細胞の浸潤・転移におけるタンパク質の相互作用と動態の制御

  小野寺康仁, 佐邊壽孝, 佐邊壽孝  実験医学  24-  (10)  1433-1439  2006/06/20  [Not refereed][Not invited]
  
• 【分子メカニズムから解き明かす疾患のサイエンス】 癌 癌細胞の浸潤・転移におけるタンパク質の相互作用と動態の制御

  小野寺 康仁, 佐邊 壽孝  実験医学  24-  (10)  1433  -1439  2006/06  [Not refereed][Not invited]
   

  癌の転移において癌細胞の浸潤活性は最も重要な性質の1つである.癌細胞は,既存分子の相互作用を改変して細胞-細胞間接着や細胞-基質間接着,運動性や細胞外基質分解活性を巧みに制御し,浸潤・転移を行うものと考えられる.これらの過程に特異的に関与するタンパク質間相互作用の同定は,正常組織への副作用を抑えた浸潤・転移阻害薬の開発において有効な情報を与えることが期待される(著者抄録)
• Breast cancer invasiveness: How does it emerge?

  HASHIMOTO SHIGERU, ONODERA YASUHITO  蛋白質 核酸 酵素  51-  (6)  803-810,496  2006/05/10  [Not refereed][Not invited]
  
• 乳がん浸潤能獲得過程 (細胞骨格と接着) -- (疾患)

  橋本 茂, 小野寺 康仁  蛋白質核酸酵素  51-  (6)  803  -810,496  2006/05  [Not refereed][Not invited]
  
• 浸潤性乳癌細胞のAMAP1/コータクチン相互作用インターフェースの癌浸潤阻害剤の標的としての評価

  橋本 茂, 廣瀬 まゆみ, 橋本 あり, 森重 真毅, 山田 敦子, 小野寺 康仁, 小川 栄治, 和田 洋巳, 池上 貴久, 中川 敦史, 佐邊 壽孝  日本癌学会総会記事  64回-  309  -309  2005/09  [Not refereed][Not invited]
  
• 浸潤・転移・血管新生研究の進歩 乳癌における浸潤形質獲得過程

  佐邊 壽孝, 小野寺 康仁, ナム・ジンミン, 橋本 あり, 森重 真毅, 橋本 茂  日本癌学会総会記事  64回-  513  -513  2005/09  [Not refereed][Not invited]
  
• 細胞間接触によるArf6の負の制御

  三浦浩一, 小野寺康仁, 佐辺寿孝  日本癌学会学術総会記事  64th-  167  2005/08/15  [Not refereed][Not invited]
  
• Arf6ユビキチン化機構と乳癌細胞浸潤性獲得過程との関連性

  矢野元, 橋本茂, 小野寺康仁, 小野寺康仁, 佐辺寿孝, 佐辺寿孝  日本癌学会学術総会記事  64th-  173  2005/08/15  [Not refereed][Not invited]
  
• 乳癌細胞におけるArf6蛋白質の量的制御機構と浸潤性獲得

  矢野 元, 古林 格, 小野寺 康仁, 橋本 茂, 佐邊 壽孝  日本癌学会総会記事  63回-  265  -265  2004/09  [Not refereed][Not invited]
  
• Arf6は乳癌細胞の浸潤性獲得において必須の分子装置の一つである

  橋本 茂, 小野寺 康仁, 橋本 あり, 森重 真毅, 田中 美和, 浜口 道成, 佐邊 壽孝  日本癌学会総会記事  63回-  265  -265  2004/09  [Not refereed][Not invited]
  
• 浸潤性乳癌細胞におけるAMAP1の発現と機能

  小野寺 康仁, 橋本 茂, 真崎 雄一, 橋本 あり, 森重 真毅, 松浦 成昭, 佐邊 壽孝  日本癌学会総会記事  63回-  267  -267  2004/09  [Not refereed][Not invited]
  

Research Grants & Projects

• 放射線治療効果向上を目指した新規細胞標的～インフルエンサー・セルの探索～

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2022/04 -2026/03 

  Author : Nam JinMin, 小野寺 康仁, 白土 博樹, 清水 伸一
• Optimization of cancer treatment based on intra-tumor metabolic geometry

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

  Date (from‐to) : 2021/10 -2026/03 

  Author : 小野寺 康仁, 西岡 蒼一郎, Nam JinMin

   

  本年度は、腫瘍内の代謝微小環境をより適切に再現するためのin vitro解析系の開発に着手した。超高線量率照射治療（FLASH-RT）による正常組織の保護作用においては、酸素濃度の急激な低下が関与していると考えられている。そこで、三次元培養系において組織内酸素濃度を適切に調整するための条件設定を試みた。酸素濃度により燐光が変化するプローブを用いて、三次元ゲルの高さ（厚み）方向の位置による酸素濃度への影響を検討した。また、酸素とグルコース濃度の同時調整を想定して、三次元培養系における「細胞特異的糖代謝制御法」の有用性の確認を行った。この方法では、特定の細胞にのみグルコースを供給するために特殊な「グルコース前駆体」を用いるが、当該物質は酸素と同様に三次元ゲルによって拡散が影響され得るため、三次元ゲル深部における十分なグルコース供給については検討の余地が残っていた。そこで三次元ゲル深部に培養したがん細胞の生存性や悪性形質を十分に担保できることを指標として、「細胞特異的糖代謝制御法」の適用可能性についての解析を行った。上述の「グルコース前駆体」として用いることができる物質は複数の種類が存在し、細胞内での代謝効率や、細胞への添加可能な濃度などがそれぞれ異なっている。それぞれの物質について、上記の目的における適性や、必要な添加濃度などの検討を行った。その結果、少なくとも一つについては十分な効率での「グルコース供給」が可能であることを確認した。 なお、本課題と関連のある業績として、腫瘍内代謝により起こる微小環境の酸性化に関する論文を発表した。その過程で、本課題においても有用な、細胞内外のpH測定を行うための実験系を確立することができた。
• Research and development about helium and proton hybrid system with proton CT and Immuno-radiotherapy

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

  Date (from‐to) : 2019/04 -2024/03 

  Author : 白土 博樹, 宮本 直樹, 高尾 聖心, 茶本 健司, 橋本 孝之, Nam JinMin, 西岡 健太郎, 小野寺 康仁, 松浦 妙子, 梅垣 菊男, 平田 雄一, 田中 創大, 清水 伸一

   

  ① 2019年度に決定した、短時間（0.1秒以下）でエネルギー変更可能な小型加速器の基本設計に基づき、小型加速器の要素技術の設計を行い、短時間（0.1秒以下）でエネルギー変更を可能とする回転ガントリーを含む照射・輸送系の磁場制御設計を行った。 ② 陽子からヘリウムに短時間で加速粒子を変更できる混合加速方式を検討、2023年度に制作開始するべく、陽子線CTの機器としての仕様を検討した。 ③ 陽子線CT値-ヘリウムSPR変換プロセスと、X線CT値利用時の精度を比較し、高エネルギー陽子線CTに必要な要素機器と制御方式の仕様を明確化した。 ④ 高エネルギー陽子線ビームを照射する場合に、ビームの人体への入射方向を意図的に偏心させ、ノズルの外側から照射野中心に向かったビームアングルとするための加速器・照射系の検討を行った。 ⑤ PD-1阻害剤およびPD-L1阻害剤を用いて、がん細胞の制御に最適なLET、ROSとミトコンドリアの分布を計測し、放射線と阻害剤の組み合わせによる相関を検討した。T細胞のPD-1阻害に関する条件検討を行っていたところ、当初の想定に反し、がん細胞での観察と類似の条件ではT細胞の観察が困難であることが判明した。T細胞を観察した上で条件決定することが不可欠であるため、T細胞のPD-1阻害に関する条件決定のための追加検討事項として、培養条件や使用する蛍光色素の種類を複数追加し、T細胞を観察するための至適条件の検討を行った。
• The development of methods to control enzyme activity using protein-protein splicing and virus-like particles

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

  Date (from‐to) : 2021/07 -2023/03 

  Author : 小野寺 康仁

   

  初年度は、レンチウイルスの仕組みを利用した「ウイルス様粒子（virus-like particle, VLP）」を細胞外からのタンパク質導入法として用いて、これにタンパク質トランススプライシングを媒介するsplit-inteinを組み合わせることで、ピューロマイシン代謝酵素（puromycin N-acetyltransferase, PAC）の活性制御を試みた。レンチウイルスはヒト免疫不全ウイルス（HIV）のgag-pol（gp）遺伝子を用いているが、これにC末端側split-intein（IntC）を付加したPACのC末端側断片（PACC）を融合し、接続部分にはHIVプロテアーゼによる切断配列を挿入した。エンベロープタンパク質であるヒト水疱性口内炎ウイルスのGタンパク質（vesicular stomatitis virus G-protein, VSV-G）をコードするプラスミドDNAと、上記の改変HIV gpをコードするプラスミドDNAを同時に細胞内導入し、得られたVLPを単離したところ、その内部においてIntC-PACCがHIV gpより切り出されていることを確認した。また、N末端側split-intein（IntN）を付加したPACのN末端側断片（PACN）であるPACN-IntNを発現する細胞に上記のVLPを添加するとピューロマイシンへの耐性が生じることを確認した。以上の成果は国際学術誌において報告した。
• Analysis of cell-to-cell communication to identify novel targets of radiotherapy

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : Nam Jin-Min

   

  Radiation therapy is widely used in cancer treatment, but understanding of the radiation effect on cancer cells is essential to improve therapeutic efficacy. To elucidate the molecular mechanisms of radiotherapy resistance mediated by these pathways, we analyzed vesicular trafficking pathways that specifically regulate factors such as extracellular vesicles secreted from cancer cells after irradiation. In this study, our data suggested that the secreted epiregulin into the extracellular space via Rab27b pathway is involved in radiotherapy resistance of glioblastoma cells.
• Analysis of the mechanisms by which intra-tumor metabolic microenvironment leads to radioresistance

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

  Date (from‐to) : 2018/04 -2021/03 

  Author : Onodera Yasuhito

   

  Using cell-specific regulation of glucose metabolism, we co-cultured glucose-sufficient and glucose-deficient cancer cells and established "metabolic cooperation" between them to analyze viability, intracellular metabolism, phenotypic changes, and response to radiation. The above experimental system revealed that cancer cells in a glucose-depleted state maintain their viability by obtaining metabolites from glucose-sufficient cells, and that oxidative stress and ER stress in the former cells are markedly suppressed. Estimation of the mediating substances and mechanisms, as well as the various phenotypic changes that occur in the metabolic cooperative state, are currently under analysis.
• Development of intracellular binary counter for recording the gene expression change

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

  Date (from‐to) : 2016/04 -2019/03 

  Author : Onodera Yasuhito

   

  In this study, we sought to develop a set of genetic constructions which records the change of the expression (ON/OFF) of the gene of interest in each cell. In order to record as many times of the expression change as possible, we aimed for the construction of the binary counter. For the proper function, these constructs require the single copy integration into the target cell genome, which is currently achieved by genome editing followed by single cell cloning. In these processes, however, heterogeneity of the cells, which is also a primary subject of this study, will be inevitably compromised. In particular, for the analyses of cancer cells, loss of heterogeneity may lead to contradictory results. Therefore, we also sought to establish a novel genetic markers by which single copy integration of multiple genetic constructs can be easily achieved.
• Radiosensitization by mitochondrial ROS amplification

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

  Date (from‐to) : 2015/04 -2018/03 

  Author : Onodera Yasuhito, NAM Jin-Min

   

  Arf6 and AMAP1 are highly expressed in different cancers including breast cancer, and mediate integrin recycling to plasma membrane to promote cancer invasion and metastasis. On the other hand, signaling downstream of integrins is well known to contribute to resistance to radiation and chemotherapy. In this study, we have shown that reduction of the integrin signaling by blockade of the Arf6-AMAP1 pathway inhibits intracellular mitochondrial distribution, resulting in aggregated mitochondria around the nucleus, which induces amplification of the reactive oxygen species (ROS) produced by ionizing radiation. We have also revealed molecules involved in the regulation of mitochondrial distribution downstream of integrins, and how they interact with each other. Further analyses may lead to the establishment of a new modality for radio-sensitization, which is based on the ROS amplification mediated by mitochondrial aggregation.
• Possible molecular mechanisms of normal cells and invasive breast cancer cells after radiation treatment

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

  Date (from‐to) : 2013/04 -2016/03 

  Author : NAM JINMIN, ONODERA YASUHITO

   

  In this study, we investigated molecular mechanisms to improve the radiation therapy by targeting specific molecules which are involved in not only preservation of the functions in normal cell structure but also suppressing tumor growth, recurrence and metastasis. To find candidate genes, we performed microarray analysis in a mammary epithelial cell line which has basal polarity, or invasive breast cancer cell lines in three-dimensional laminin rich extracellular matrix (3D lrECM). We found that β1-integrin and its downstream molecules are involved in the disruption of basal polarity structure and invasive transition on non-malignant mammary epithelial cells in 3D lrECM. In addition, we also found that up-regulated integrin expression on the cell surface may have important roles in the invasive activity after radiation treatment in breast cancer cells.
• Regulation of interaction between cells and microenvironment by metabolic cooperation

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

  Date (from‐to) : 2013 -2015 

  Author : Onodera Yasuhito

   

  We have previously reported that regulation of glucose metabolism is tightly connected to that of signaling cascades, which is fundamental for the establishment and maintenance of tissue architecture. In order to examine whether and how metabolic cooperation between different types of cells regulates the cell-cell and cell-microenvironment interaction and orchestrates cell behaviors, we have investigated new techniques to obtain double-layered structure made of different types of cells, which resembles acinar and tubular structures of mammary gland. We have also established a novel method to incorporate stable isotope-labeled glucose (e.g. 13C glucose) into only specific cells, which enables us to examine and manipulate metabolic cooperation between different types of cells in the microenvironment. Using these methods, the metabolic cooperation between luminal epithelial and myoepithelial cells of mammary gland is now investigated.
• Analysis of the molecular mechanism of radiation induced invasiveness

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

  Date (from‐to) : 2011 -2012 

  Author : NAM JinMin, ONODERA Yasuhito

   

  Radiation therapy is the common treatment for breast cancer, and the primary goal of therapy is to prevent invasive recurrence. The detailed molecular mechanism of invasive recurrence after radiation therapy is not well understood. This study investigated the effect of radiation on a three-dimensional culture model of non-invasive breast cancer, and identified important molecule and possible molecular mechanism underlying emergence of invasiveness. Our results suggest that α5ss1-integrin via NF-κB signaling is an important mediator of invasive progression after radiation treatment on non-invasive breast cancer cells in three-dimensional extracellular matrix.
• Research for Real-time Tracking Medical Science

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

  Date (from‐to) : 2009 -2012 

  Author : SHIRATO Hiroki, HONMA Sato, TAMAKI Nagara, KUGE Yuji, MIZUTA Masahiro, DATE Hiroyuki, TANAKA Masaki, HAGA Hisashi, NISHIOKA Takeshi, KATO Motothugu, CHAMOTO Kenji, OIZUMI Satoshi, MATSUNAGA Naofumi, OKIMOTO Tomoaki, HAYAKAWA Kazushige, NISHIO Teiji, TADANO Shigeru, ISHIKAWA Masayori, ONODERA Yasuhito, SHIBUYA Keiko, HAMADA Toshiyuki, ONIMARU Rikiya, SHIMIZU Shinichi, TSUCHIYA Kazuhiko, KATOH Norio, KINOSHITA Rumiko, INOUE Tetsuya, ONODERA Syunsuke, TAKAO Seishin, KAGA Kichizo, TERAE Satoshi, ONODERA Yuya, SAKUHARA Yusuke, MANABE Noriko, ABO Daisuke, KATO Fumi, KHIN KHIN Tha, NAM Jin-min, SABE Hisataka, INUBUSHI Masayuki, SHINAGAWA Naofumi, KENNETH Sutherland

   

  Up to now, in the field of basic medicine from micro-level to animal level, to track and quantify the three-dimensional spatial information along the time axis in real-time has not caught up with the accuracy in the field of clinical medicine. By handing big data of “motion in life, a new idea of real-time radiotherapy and a new precise four-dimensional computed tomography has been developed based on the data of actual tumor motion in the human body.
• Molecular mechanisms by which increased glucose metabolism regulates signaling pathways in cancer

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

  Date (from‐to) : 2010 -2011 

  Author : ONODERA Yasuhito

   

  In this study, using three dimensional culture of breast cancer cells, the principal investigator examined mechanisms by which increased glucose metabolism regulates signaling pathways in cancer cells. In particular, the PI focused on hexosamine biosynsethis pathway(HBP), which branches from glycolytic pathway, and found that the enzymes in HBP play fundamental roles in signaling activity and malignant phenotype in cancer cells. Moreover, O-GlcNAc modification of proteins, which is downstream of HBP, and the enzyme responsible for O-GlcNAc modification were also found to be essential. Target proteins modified with O-GlcNAc specifically in cancer cells were also identified, and the roles of the proteins and the modification are now under investigation.

Educational Activities

Teaching Experience