Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Biochemistry

Affiliation (Master)

• Faculty of Medicine　Physiological Science　Biochemistry

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Profile and Settings

Degree

• Doctor of Philosophy（Kansai Medical University）
• Master of Science（Kobe University）

Profile and Settings

• Name (Japanese)

  Hashimoto

• Name (Kana)

  Ari

• Name

  200901058984667481

Achievement

Research Interests

• Arf6   AMAP1   ArfGEF   乳癌   癌浸潤   血管新生   細胞浸潤   細胞内輸送   ノックアウトマウス   endocytosis   細胞運動   ArfGAP   AMAP2   分子生物学   Molecular Biology   

Research Areas

• Life sciences / Medical biochemistry
• Life sciences / Cell biology

Research Experience

• 2009/10 - Today 北海道大学 医学(系)研究科(研究院) 助教
• 2002/04 - 2009/09 大阪バイオサイエンス研究所 研究員
• 小野薬品工業 主任研究員

Published Papers

• MitoNEET reduces the mitochondrial oxidative phosphorylation during epithelial-mesenchymal transition

  Haruka Handa, Yasuhito Onodera, Tsukasa Oikawa, Shingo Takada, Koji Ueda, Daiki Setoyama, Takashi Yokota, Miwako Yamasaki, Masahiko Watanabe, Yoshizuki Fumoto, Ari Hashimoto, Soichiro Hata, Masaaki Murakami, Hisataka Sabe

  2024/07/29 

  Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions
• p53 ensures the normal behavior and modification of G1/S-specific histone H3.1 in the nucleus

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

  Life Science Alliance 7 (9) e202402835  2024/06 [Refereed][Not invited]
  
• ARF6-AMAP1経路は免疫抑制性ケモカインの発現を誘導し、免疫回避に有利に機能する(ARF6-AMAP1 pathway is linked to induction of immunosuppressive chemokine expression for favor immune evasion)

  橋本 あり, 半田 悠, 畑 宗一郎, 奥崎 大介, 麓 佳月, 蔦保 暁生, 西川 義浩, 児玉 裕三, 平野 聡, 橋本 茂, 佐邊 壽孝

  日本癌学会総会記事 82回 1922 - 1922 0546-0476 2023/09
• ARF6-AMAP1経路は免疫抑制性ケモカインの発現を誘導し、免疫回避に有利に機能する(ARF6-AMAP1 pathway is linked to induction of immunosuppressive chemokine expression for favor immune evasion)

  橋本 あり, 半田 悠, 畑 宗一郎, 奥崎 大介, 麓 佳月, 蔦保 暁生, 西川 義浩, 児玉 裕三, 平野 聡, 橋本 茂, 佐邊 壽孝

  日本癌学会総会記事 82回 1922 - 1922 0546-0476 2023/09
• 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
• A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling.

  Taiga Maemoto, Yuichi Kitai, Runa Takahashi, Haruka Shoji, Shunsuke Yamada, Shiho Takei, Daiki Ito, Ryuta Muromoto, Jun-Ichi Kashiwakura, Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Toyoyuki Ose, Kenji Oritani, Tadashi Matsuda

  The Journal of biological chemistry 299 (1) 102724 - 102724 2022/11/18 

  Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anti-cancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.
• Tumor-derived interleukin-34 creates an immunosuppressive and chemoresistant tumor microenvironment by modulating myeloid-derived suppressor cells in triple-negative breast cancer.

  Nabeel Kajihara, Takuto Kobayashi, Ryo Otsuka, Junko Nio-Kobayashi, Tomohiro Oshino, Masato Takahashi, Seiichi Imanishi, Ari Hashimoto, Haruka Wada, Ken-Ichiro Seino

  Cancer immunology, immunotherapy : CII 2022/09/14 [Refereed]
   

  Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by a lack of therapeutic targets. The paucity of effective treatment options motivated a number of studies to tackle this problem. Immunosuppressive cells infiltrated into the tumor microenvironment (TME) of TNBC are currently considered as candidates for new therapeutic targets. Myeloid-derived suppressor cells (MDSCs) have been reported to populate in the TME of TNBC, but their roles in the clinical and biological features of TNBC have not been clarified. This study identified that interleukin-34 (IL-34) released by TNBC cells is a crucial immunomodulator to regulate MDSCs accumulation in the TME. We provide evidence that IL-34 induces a differentiation of myeloid stem cells into monocytic MDSCs (M-MDSCs) that recruits regulatory T (Treg) cells, while suppressing a differentiation into polymorphonuclear MDSCs (PMN-MDSCs). As a result, the increase in M-MDSCs contributes to the creation of an immunosuppressive TME, and the decrease in PMN-MDSCs suppresses angiogenesis, leading to an acquisition of resistance to chemotherapy. Accordingly, blockade of M-MDSC differentiation with an estrogen receptor inhibitor or anti-IL-34 monoclonal antibody suppressed M-MDSCs accumulation causing retardation of tumor growth and restores chemosensitivity of the tumor by promoting PMN-MDSCs accumulation. This study demonstrates previously poorly understood mechanisms of MDSCs-mediated chemoresistance in the TME of TNBC, which is originated from the existence of IL-34, suggesting a new rationale for TNBC treatment.
• Central Roles of STAT3-Mediated Signals in Onset and Development of Cancers: Tumorigenesis and Immunosurveillance.

  Shigeru Hashimoto, Ari Hashimoto, Ryuta Muromoto, Yuichi Kitai, Kenji Oritani, Tadashi Matsuda

  Cells 11 (16) 2022/08/22 [Refereed]
   

  Since the time of Rudolf Virchow in the 19th century, it has been well-known that cancer-associated inflammation contributes to tumor initiation and progression. However, it remains unclear whether a collapse of the balance between the antitumor immune response via the immunological surveillance system and protumor immunity due to cancer-related inflammation is responsible for cancer malignancy. The majority of inflammatory signals affect tumorigenesis by activating signal transducer and activation of transcription 3 (STAT3) and nuclear factor-κB. Persistent STAT3 activation in malignant cancer cells mediates extremely widespread functions, including cell growth, survival, angiogenesis, and invasion and contributes to an increase in inflammation-associated tumorigenesis. In addition, intracellular STAT3 activation in immune cells causes suppressive effects on antitumor immunity and leads to the differentiation and mobilization of immature myeloid-derived cells and tumor-associated macrophages. In many cancer types, STAT3 does not directly rely on its activation by oncogenic mutations but has important oncogenic and malignant transformation-associated functions in both cancer and stromal cells in the tumor microenvironment (TME). We have reported a series of studies aiming towards understanding the molecular mechanisms underlying the proliferation of various types of tumors involving signal-transducing adaptor protein-2 as an adaptor molecule that modulates STAT3 activity, and we recently found that AT-rich interactive domain-containing protein 5a functions as an mRNA stabilizer that orchestrates an immunosuppressive TME in malignant mesenchymal tumors. In this review, we summarize recent advances in our understanding of the functional role of STAT3 in tumor progression and introduce novel molecular mechanisms of cancer development and malignant transformation involving STAT3 activation that we have identified to date. Finally, we discuss potential therapeutic strategies for cancer that target the signaling pathway to augment STAT3 activity.
• Orchestration of mesenchymal plasticity and immune evasiveness via rewiring of the metabolic program in pancreatic ductal adenocarcinoma.

  Ari Hashimoto, Haruka Handa, Soichiro Hata, Shigeru Hashimoto

  Frontiers in oncology 12 1005566 - 1005566 2022 

  Pancreatic ductal adenocarcinoma (PDAC) is the most fatal cancer in humans, due to its difficulty of early detection and its high metastatic ability. The occurrence of epithelial to mesenchymal transition in preinvasive pancreatic lesions has been implicated in the early dissemination, drug resistance, and cancer stemness of PDAC. PDAC cells also have a reprogrammed metabolism, regulated by driver mutation-mediated pathways, a desmoplastic tumor microenvironment (TME), and interactions with stromal cells, including pancreatic stellate cells, fibroblasts, endothelial cells, and immune cells. Such metabolic reprogramming and its functional metabolites lead to enhanced mesenchymal plasticity, and creates an acidic and immunosuppressive TME, resulting in the augmentation of protumor immunity via cancer-associated inflammation. In this review, we summarize our recent understanding of how PDAC cells acquire and augment mesenchymal features via metabolic and immunological changes during tumor progression, and how mesenchymal malignancies induce metabolic network rewiring and facilitate an immune evasive TME. In addition, we also present our recent findings on the interesting relevance of the small G protein ADP-ribosylation factor 6-based signaling pathway driven by KRAS/TP53 mutations, inflammatory amplification signals mediated by the proinflammatory cytokine interleukin 6 and RNA-binding protein ARID5A on PDAC metabolic reprogramming and immune evasion, and finally discuss potential therapeutic strategies for the quasi-mesenchymal subtype of PDAC.
• Inhibition of mutant KRAS-driven overexpression of ARF6 and MYC by an eIF4A inhibitor drug improves the effects of anti-PD-1 immunotherapy for pancreatic cancer

  Ari Hashimoto, Haruka Handa, Soichiro Hata, Akio Tsutaho, Takao Yoshida, Satoshi Hirano, Shigeru Hashimoto, Hisataka Sabe

  Cell Communication and Signaling 19 (1) 2021/12 [Refereed]
   

  Many clinical trials are being conducted to clarify effective combinations of various drugs for immune checkpoint blockade (ICB) therapy. However, although extensive studies from multiple aspects have been conducted regarding treatments for pancreatic ductal adenocarcinoma (PDAC), there are still no effective ICB-based therapies or biomarkers for this cancer type. A series of our studies have identified that the small GTPase ARF6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) are often overexpressed in different cancers, including PDAC, and closely correlate with poor patient survival. Mechanistically, the ARF6-AMAP1 pathway drives cancer cell invasion and immune evasion, via upregulating β1-integrins and PD-L1, and downregulating E-cadherin, upon ARF6 activation by external ligands. Moreover, the ARF6-AMAP1 pathway enhances the fibrosis caused by PDAC, which is another barrier for ICB therapies. KRAS mutations are prevalent in PDACs. We have shown previously that oncogenic KRAS mutations are the major cause of the aberrant overexpression of ARF6 and AMAP1, in which KRAS signaling enhances eukaryotic initiation factor 4A (eIF4A)-dependent ARF6 mRNA translation and eIF4E-dependent AMAP1 mRNA translation. MYC overexpression is also a key pathway in driving cancer malignancy. MYC mRNA is also known to be under the control of eIF4A, and the eIF4A inhibitor silvestrol suppresses MYC and ARF6 expression. Using a KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre), we here demonstrate that inhibition of the ARF6-AMAP1 pathway by shRNAs in cancer cells results in therapeutic synergy with an anti-PD-1 antibody in vivo; and furthermore, that silvestrol improves the efficacy of anti-PD-1 therapy, whereas silvestrol on its own promotes tumor growth in vivo. ARF6 and MYC are both essential for normal cell functions. We demonstrate that silvestrol substantially mitigates the overexpression of ARF6 and MYC in KRAS-mutated cells, whereas the suppression is moderate in KRAS-intact cells. We propose that targeting eIF4A, as well as mutant KRAS, provides novel methods to improve the efficacy of anti-PD-1 and associated ICB therapies against PDACs, in which ARF6 and AMAP1 overexpression, as well as KRAS mutations of cancer cells are biomarkers to identify patients with drug-susceptible disease. The same may be applicable to other cancers with KRAS mutations.
• Arid5a Promotes Immune Evasion by Augmenting Tryptophan Metabolism and Chemokine Expression.

  Gyanu Parajuli, Murat Tekguc, James B Wing, Ari Hashimoto, Daisuke Okuzaki, Takeshi Hirata, Atsushi Sasaki, Takahide Itokazu, Haruka Handa, Hirokazu Sugino, Yoshihiro Nishikawa, Hozaifa Metwally, Yuzo Kodama, Shinya Tanaka, Hisataka Sabe, Toshihide Yamashita, Shimon Sakaguchi, Tadamitsu Kishimoto, Shigeru Hashimoto

  Cancer immunology research 9 (8) 862 - 876 2021/08 [Refereed]
   

  The acquisition of mesenchymal traits leads to immune evasion in various cancers, but the underlying molecular mechanisms remain unclear. In this study, we found that the expression levels of AT-rich interaction domain-containing protein 5a (Arid5a), an RNA-binding protein, were substantially increased in mesenchymal tumor subtypes. The deletion of Arid5a in tumor cell lines enhanced antitumor immunity in immunocompetent mice, but not in immunodeficient mice, suggesting a role for Arid5a in immune evasion. Furthermore, an Arid5a-deficient tumor microenvironment was shown to have robust antitumor immunity, as manifested by suppressed infiltration of granulocytic myeloid-derived suppressor cells and regulatory T cells. In addition, infiltrated T cells were more cytotoxic and less exhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2 mRNAs and augmented their expression, resulting in enhanced tryptophan catabolism and an immunosuppressive tumor microenvironment. Thus, our findings demonstrate the role of Arid5a beyond inflammatory diseases and suggest Arid5a as a promising target for the treatment of immunotolerant malignant tumors.See related Spotlight by Van den Eynde, p. 854.
• ARF6のエフェクター分子AMAP1の高発現はPD-L1及び線維化の亢進に関わる

  橋本 あり, 蔦保 暁生, 橋本 茂, 畑 宗一郎, 加地 紫苑, 平野 聡, 佐邊 壽孝

  日本癌学会総会記事 79回 PJ14 - 2 0546-0476 2020/10
• 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
• 膵臓癌のPD-L1発現と線維化におけるARF6-AMAP1経路の役割

  蔦保 暁生, 橋本 あり, 橋本 茂, 佐邊 壽孝, 平野 聡

  日本外科学会定期学術集会抄録集 (一社)日本外科学会 120回 SF - 7 2020/08
• High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer.

  Akio Tsutaho, Ari Hashimoto, Shigeru Hashimoto, Soichiro Hata, Shion Kachi, Satoshi Hirano, Hisataka Sabe

  Cell communication and signaling : CCS 18 (1) 101 - 101 2020/06/24 [Refereed]
   

  BACKGROUND: Not merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis. METHODS: Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed. RESULTS: Elevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients. CONCLUSIONS: Together with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK. Video abstract.
• 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
• 膵癌ドライバー変異はmRNA翻訳と蛋白質プレニル化を介しARF6が駆動する癌免疫回避を促進する(Pancreatic KRAS/TP53 mutations promote ARF6-based immune evasion via activating mRNA translation and protein prenylation)

  橋本 あり, 橋本 茂, 古川 聖太郎, 蔦保 暁生, 小野寺 康人, 半田 悠, 及川 司, 水上 裕輔, 西川 義浩, 児玉 裕三, 村上 正晃, 平野 聡, 佐邊 壽孝

  日本癌学会総会記事 78回 P - 3033 0546-0476 2019/09
• 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.
• Endothelin type B receptor interacts with 78-kDa glucose-regulated protein

  Mazaki Y, Higashi T, Onodera Y, Nam JM, Hashimoto A, Hashimoto S, Horinouchi T, Miwa S

  FEBS Lett 593 (6) 644 - 651 2019/03 [Refereed][Not invited]
  
• 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.
• Epithelial-specific histone modification of the miR-96/182 locus targeting AMAP1 mRNA predisposes p53 to suppress cell invasion in epithelial cells.

  Handa H, Hashimoto A, Hashimoto S, Sugino H, Oikawa T, Sabe H

  Cell communication and signaling : CCS 16 (1) 94 - 94 2018/12 [Refereed][Not invited]
   

  BACKGROUND: TP53 mutations in cancer cells often evoke cell invasiveness, whereas fibroblasts show invasiveness in the presence of intact TP53. AMAP1 (also called DDEF1 or ASAP1) is a downstream effector of ARF6 and is essential for the ARF6-driven cell-invasive phenotype. We found that AMAP1 levels are under the control of p53 (TP53 gene product) in epithelial cells but not in fibroblasts, and here addressed that molecular basis of the epithelial-specific function of p53 in suppressing invasiveness via targeting AMAP1. METHODS: Using MDA-MB-231 cells expressing wild-type and p53 mutants, we identified miRNAs in which their expression is controlled by normal-p53. Among them, we identified miRNAs that target AMAP1 mRNA, and analyzed their expression levels and epigenetic statuses in epithelial cells and nonepithelial cells. RESULTS: We found that normal-p53 suppresses AMAP1 mRNA in cancer cells and normal epithelial cells, and that more than 30 miRNAs are induced by normal-p53. Among them, miR-96 and miR-182 were found to target the 3'-untranslated region of AMAP1 mRNA. Fibroblasts did not express these miRNAs at detectable levels. The ENCODE dataset demonstrated that the promoter region of the miR-183-96-182 cistron is enriched with H3K27 acetylation in epithelial cells, whereas this locus is enriched with H3K27 trimethylation in fibroblasts and other non-epithelial cells. miRNAs, such as miR-423, which are under the control of p53 but not associated with AMAP1 mRNA, demonstrated similar histone modifications at their gene loci in epithelial cells and fibroblasts, and were expressed in these cells. CONCLUSION: Histone modifications of certain miRNA loci, such as the miR-183-96-182 cistron, are different between epithelial cells and non-epithelial cells. Such epithelial-specific miRNA regulation appears to provide the molecular basis for the epithelial-specific function of p53 in suppressing ARF6-driven invasiveness.
• Frequent overexpression of AMAP1, an Arf6 effector in cell invasion, is characteristic of the MMTV-PyMT rather than the MMTV-Neu human breast cancer model

  Yutaro Otsuka, Tsukasa Oikawa, Hinako Yoshino, Shigeru Hashimoto, Haruka Handa, Hiroki Yamamoto, Ari Hashimoto, Hisataka Sabe

  Cell Communication and Signaling 16 (1) 1  1478-811X 2018/01/05 [Refereed][Not invited]
   

  Background: The small GTPase Arf6 and its downstream effector AMAP1 (also called ASAP1/DDEF1) constitute a signaling pathway promoting cell invasion, in which AMAP1 interacts with several different proteins, including PRKD2, EPB41L5, paxillin, and cortactin. Components of this pathway are often overexpressed in human breast cancer cells, to be correlated with poor prognosis of the patients, whereas overexpression of the Arf6 pathway did not correlate with the four main molecular classes of human breast tumors. In this pathway, receptor tyrosine kinases, including EGFR and Her2, activate Arf6 via GEP100. MMTV-PyMT mice and MMTV-Neu mice are well-established models of human breast cancer, and exhibit the early dissemination and the lung metastasis, by utilizing protein tyrosine phosphorylation for oncogenesis. PyMT-tumors and Neu-tumors are known to have overlapping gene expression profiles, which primarily correspond to the luminal B-type of human mammary tumors, although they differ in the time necessary for tumor onset and metastasis. Given the common usage of protein tyrosine phosphorylation, as well as the frequent use of these animal models for studying breast cancer at the molecular level, we here investigated whether mammary tumors in these mouse models utilize the Arf6-based pathway for invasion. Methods: Expression levels of Arf6, AMAP1, and GEP100 were analyzed in PyMT-tumors and Neu-tumors by western blotting. Expression of Arf6 and AMAP1 was also analyzed by immunohistochemistry. The involvement of AMAP1 in invasion, and the possible correlation of its high expression levels with cancer mesenchymal properties were also investigated. Results: We found that PyMT-tumors, but not Neu-tumors, frequently overexpress AMAP1 and use it for invasion, whereas both types of tumors expressed Arf6 and GEP100 at different levels. High levels of the AMAP1 expression among PyMT-tumor cells were frequently correlated with loss of the epithelial marker CK8 and also with expression of the mesenchymal marker vimentin both at the primary sites and at sites of the lung metastases. Conclusions: PyMT-tumors appear to frequently utilize the Arf6-based invasive machinery, whereas Neu-tumors do not. Our results suggest that MMTV-PyMT mice, rather than MMTV-Neu mice, are useful to study the Arf6-based mammary tumor malignancies, as a representative model of human breast cancer.
• 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.
• Prognostic significance of erythrocyteproteinband 4.1-like5 expression in upper urinary tract urothelial carcinoma

  Tatsuaki Daimon, Takeo Kosaka, Eiji Kikuchi, Shuji Mikami, Yasumasa Miyazaki, Ari Hashimoto, Shigeru Hashimoto, Ryuichi Mizuno, Akira Miyajima, Yasunori Okada, Hisataka Sabe, Mototsugu Oya

  UROLOGIC ONCOLOGY-SEMINARS AND ORIGINAL INVESTIGATIONS 35 (9) 543.e17 - 543.e24 1078-1439 2017/09 [Refereed][Not invited]
   

  Objectives: The erythrocyte protein band 4.1-like5 (EPB4.1L5) regulates E-cadherin in cancer invasion and metastasis inducing epithelial-to-mesenchymal transition. This study aimed to investigate the biological significance of EPB4.1L5 in upper urinary tract urothelial carcinoma (UTUC). Methods: Retrospective analysis of the clinical records of 165 patients with UTUC (Ta-4NOMO) subjected to radical nephroureterectomy and immunohistochemical examination of EPB4.1L5 expression in those tissues. Results: The median follow-up period was 62.2 months (interquartile range = 77.0). The score of EPB4.1L5 significantly correlated with tumor grade, pathological T stage, and lymphovascular invasion (all P < 0.001). The 5-year Kaplan-Meier recurrence-free survival and cancer-specific survival rates were 54.1% and 59.5% in patients with high EPB4.1L5 expression, compared with 81.6% and 87.2%, (all P < 0.001) in their counterparts. Multivariate analyses revealed that high expression of EPB4.1L5 was one of the independent prognostic factors for tumor recurrence (P = 0.022, HR = 2.40) and cancer-specific survival (P = 0.015, HR = 2.94). Conclusion: High EPB4.1L5 expression was related to worse clinical outcome in patients with UTUC. These results indicated that EPB4.1L5 could provide prognostic information in patients with UTUC regarding epithelial-to-mesenchymal transition. (C) 2017 Elsevier Inc. All rights reserved.
• 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.
• Arf6 and its ZEB1-EPB41L5 mesenchymal axis are required for both mesenchymal- and amoeboid-type invasion of cancer cells.

  Handa H, Hashimoto A, Hashimoto S, Sabe H

  Small GTPases 9 (5) 1 - 7 2154-1248 2016/10 [Refereed][Not invited]
   

  Modes of cancer invasion interchange between the mesenchymal type and amoeboid type in response to the microenvironment, in which RhoA and Rac1 are selectively required to perform different modes of actin-cytoskeletal remodeling. Membrane remodeling is another integral part of invasion. Arf6 regulates the recycling of molecules at the cell periphery, and is often overexpressed in malignant cancers together with its effector AMAP1/ASAP1/DDEF1. This pathway promotes mesenchymal-type invasion when AMAP1 binds to EPB41L5, a mesenchymal-specific protein induced by ZEB1. Here we show that the Arf6-AMAP1-EPB41L5 pathway, and ZEB1, are also crucial for amoeboid-type invasion, via receptor tyrosine kinase and G-protein-coupled receptor signaling. Thus, Arf6 appears to be necessary for both RhoA- and Rac1-driven cancer invasion. Moreover, amoeboid-type cancer invasion may require the activation of some type of mesenchymal program within the cancer cells.
• ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance.

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

  Oncogenesis Nature Publishing Group 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.
• Tumor responsiveness to statins requires overexpression of the ARF6 pathway.

  Sabe H, Hashimoto A, Hashimoto S, Oikawa T

  Molecular & cellular oncology 3 (4) e1185564  2016/07 [Refereed][Not invited]
   

  The mevalonate pathway results in the prenylation of small GTPases, which are pivotal for oncogenesis and cancer malignancies. However, inhibitors of this pathway, such as statins, have not necessarily produced favorable results in clinical trials. We recently identified properties of statin responders, together with the underlying molecular mechanisms and simple biomarkers to predict these responders.
• 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.
• AMAP1 as a negative-feedback regulator of nuclear factor-kappa B under inflammatory conditions

  Dat Nguyen Tien, Masako Kishihata, Ayumu Yoshikawa, Ari Hashimoto, Hisataka Sabe, Eiichiro Nishi, Kaeko Kamei, Hidenori Arai, Toru Kita, Takeshi Kimura, Masayuki Yokode, Noboru Ashida

  SCIENTIFIC REPORTS 4 5094  2045-2322 2014/05 [Refereed][Not invited]
   

  NF-kappa B is a major transcriptional factor regulating many cellular functions including inflammation; therefore, its appropriate control is of high importance. The detailed mechanism of its activation has been well characterized, but that of negative regulation is poorly understood. In this study, we showed AMAP1, an Arf-GTPase activating protein, as a negative feedback regulator for NF-kappa B by binding with IKK beta, an essential kinase in NF-kappa B signaling. Proteomics analysis identified AMAP1 as a binding protein with IKK beta. Overexpression of AMAP1 suppressed NF-kappa B activity by interfering the binding of IKK beta and NEMO, and deletion of AMAP1 augmented NF-kappa B activity. The activation of NF-kappa B induced translocation of AMAP1 to cytoplasm from cell membrane and nucleus, which resulted in augmented interaction of AMAP1 and IKK beta. These results demonstrated a novel role of AMAP1 as a negative feedback regulator of NF-kappa B, and presented it as a possible target for anti-inflammatory treatments.
• High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients

  Hiroki Sato, Kanako C. Hatanaka, Yutaka Hatanaka, Hiromitsu Hatakeyama, Ari Hashimoto, Yoshihiro Matsuno, Satoshi Fukuda, Hisataka Sabe

  CELL COMMUNICATION AND SIGNALING 12 17  1478-811X 2014/03 [Refereed][Not invited]
   

  Background: Despite recent advances in cancer therapeutics in general, the survival of patients with head and neck squamous cell carcinomas (HNSCCs) has not improved substantially over the past few decades. HNSCC cells often exhibit invasive and metastatic phenotypes, and expression of epidermal growth factor receptor (EGFR) and cortactin has been highly implicated in the development of malignancy in HNSCCs. We have shown previously that an Arf6 pathway, in which Arf6 is activated by GEP100 and employs AMAP1 (also called DDEF1 or ASAP1) as its downstream effector, is pivotal for the invasion and metastasis of different breast cancer cells. This pathway is activated by receptor tyrosine kinases, including EGFR; and moreover, AMAP1 physically associates with cortactin, in which inhibition of this binding effectively blocks invasion and metastasis. We here investigated whether the expression of Arf6 pathway components correlates with the poor prognosis of HNSCC patients. We have shown previously that AMAP1 protein levels are not correlated with its mRNA levels, and hence we here employed immunohistochemical staining of HNSCC clinical specimens to investigate AMAP1 protein levels. Results: We found that high levels of AMAP1 protein expression on its own, as well as its co-overexpression with EGFR statistically correlates with poor disease-free survival and poor overall survival, while high levels of cortactin expression or its co-expression with EGFR did not. Conclusion: Our identification of predictive biomarkers, together with our previous findings on the coherent signaling pathway that these biomarkers ultimately generate should be powerful information for the further development of HNSCC therapeutics.
• 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.
• 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.
• Engagement of Overexpressed Her2 with GEP100 Induces Autonomous Invasive Activities and Provides a Biomarker for Metastases of Lung Adenocarcinoma

  Toshi Menju, Shigeru Hashimoto, Ari Hashimoto, Yutaro Otsuka, Haruka Handa, Eiji Ogawa, Yoshinobu Toda, Hiromi Wada, Hiroshi Date, Hisataka Sabe

  PLOS ONE 6 (9) e25301  1932-6203 2011/09 [Refereed][Not invited]
   

  Overexpression of Her2/ErbB2/Neu in cancer is often correlated with recurrent distant metastasis, although the mechanism still remains largely elusive. We have previously shown that EGFR, when tyrosine-phosphorylated, binds to GEP100/BRAG2 to activate Arf6, which induces cancer invasion and metastasis. We now show that overexpressed Her2 in lung adenocarcinoma cells also employs GEP100. Like EGFR-GEP100 binding, this association is primarily mediated by the pleckstrin homology (PH) domain of GEP100 and Tyr1139/Tyr1196 of Her2. Tyr1139/Tyr1196 are autonomously phosphorylated, when Her2 is overexpressed. Accordingly, invasive activities mediated by the Her2-GEP100 pathway are not dependent on external factors. Blocking Her2-GEP100 binding, as well as its signaling pathway all inhibit cancer invasive activities. Moreover, our clinical study indicates that co-overexpression of Her2 with GEP100 in primary lung adenocarcinomas of patients is correlated with the presence of their node-metastasis with a statistical significance. Since the GEP100 PH domain interacts with both Her2 and EGFR, targeting this domain may provide novel cancer therapeutics.
• GEP100-Arf6-AMAP1-Cortactin Pathway Frequently Used in Cancer Invasion Is Activated by VEGFR2 to Promote Angiogenesis

  Ari Hashimoto, Shigeru Hashimoto, Ryo Ando, Kosuke Noda, Eiji Ogawa, Hirokazu Kotani, Mayumi Hirose, Toshi Menju, Masaki Morishige, Toshiaki Manabe, Yoshinobu Toda, Susumu Ishida, Hisataka Sabe

  PLOS ONE 6 (8) e23359  1932-6203 2011/08 [Refereed][Not invited]
   

  Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy. Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF-or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.
• 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)
• The EGFR-GEP100-Arf6 pathway in breast cancer Full invasiveness is not from the inside

  Hisataka Sabe, Shigeru Hashimoto, Masaki Morishige, Ari Hashimoto, Eiji Ogawa

  CELL ADHESION & MIGRATION 2 (2) 71 - 73 1933-6918 2008/04 [Refereed][Not invited]
   

  Arf6 and its effector AMAP1 are overexpressed in malignant breast cancer cells, and are involved in their invasion and metastasis. We recently revealed that GEP100, a guanine nucleotide exchanging factor, is responsible for the activation of Arf6 which induces invasion and metastasis. GEP100 associated directly with ligand-activated epidermal growth factor receptor (EGFR) to be activated. Disruption of E-cadherin-mediated cell-cell adhesion is one of the major steps involved in acquisition of invasive phenotypes of most carcinomas. The EGFR-GEP100-Arf6 pathway not only activated matrix invasion activity but also perturbed E-cadherin function. GEP100 was found to be expressed in more than 80% of invasive ductal carcinomas. However, 60% of ductal carcinomas in situ were also positive for GEP100, in which GEP100 was preferentially coexpressed with EGFR in their malignant cases. Microenvionments have been highly implicated in the development of tumor malignancy. Our results reveal an aspect of the precise molecular mechanism of cancer invasion and metastasis, in which full invasiveness is not acquired just by alterations of cancer cells themselves, but their microenvironments may also play pivotal roles.
• GeP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

  Masaki Morishige, Shigeru Hashimoto, Eiji Ogawa, Yoshinobu Toda, Hirokazu Kotani, Mayumi Hirose, Shumei Wei, Ari Hashimoto, Atsuko Yamada, Hajime Yano, Yuichi Mazaki, Hiroshi Kodama, Yoshinori Nio, Toshiaki Manabe, Hiromi Wada, Hidenori Kobayashi, Hisataka Sabe

  NATURE CELL BIOLOGY 10 (1) 85 - U70 1465-7392 2008/01 [Refereed][Not invited]
   

  Epidermal growth factor (EGF) receptor ( EGFR) signalling is implicated in tumour invasion and metastasis(1,2). However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells(3-6). Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells(7) to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.
• CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery

  Nam JM, Onodera Y, Mazaki Y, Miyoshi H, Hashimoto S, Sabe H

  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.
• Neutrophil direction sensing and superoxide production linked by the GTPase-activating protein GIT2

  Y Mazaki, S Hashimoto, T Tsujimura, M Morishige, A Hashimoto, K Aritake, A Yamada, JM Nam, H Kiyonari, K Nakao, H Sabe

  NATURE IMMUNOLOGY 7 (7) 724 - 731 1529-2908 2006/07 [Refereed][Not invited]
   

  In neutrophils, superoxide anion production generally accompanies chemotaxis and functions in killing invading pathogens. The GIT2 GTPase-activating protein binds to the guanine nucleotide-exchange factor alpha PIX. Here we show that GIT2 was necessary for directional chemotaxis and for the suppression of superoxide production in G protein-coupled receptor-stimulated neutrophils. GIT2 was also necessary for the orientation of superoxide production toward chemoattractant sources. GIT2 suppressed the activity of ADP ribosylation factor 1 and was a component of the G beta gamma subunit-mediated direction-sensing machinery 'downstream' of G protein-coupled receptor signaling. This study establishes a function for GIT2 in linking chemotaxis and superoxide production in neutrophils and shows that loss of GIT2 in vivo leads to an immunodeficient state.
• [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]
  
• Targeting AMAP1 and cortactin binding bearing an atypical src horology 3/proline interface for prevention of breast cancer invasion and metastasis

  S Hashimoto, M Hiroso, A Hashimoto, M Morishige, A Yamada, H Hosaka, KI Akagi, E Ogawa, C Oneyama, T Agatsuma, M Okada, H Kobayashi, H Wada, H Nakano, T Ikegami, A Nakagawa, H Sabe

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 (18) 7036 - 7041 0027-8424 2006/05 [Refereed][Not invited]
   

  Invasive potentials of carcinomas greatly contribute to their metastasis, which is a major threat in most cancers. We have recently shown that Arf6 plays a pivotal role in breast cancer invasive activities and identified AMAP1 as an effector of GTP-Arf6 in invasion. Expression of AMAP1 correlates well with invasive phenotypes of primary tumors of the human breast. We also have shown that AMAP1 functions by forming a trimeric protein complex with cortactin and paxillin. In this complex, AMAP1 binds to the src homology 3 (SH3) domain of cortactin via its proline-rich peptide, SKKRPPPPPPGHKRT. SH3 domains are known to bind generally to the proline-rich ligands with a one-to-one stoichiometry. We found that AMAP1/cortactin binding is very atypical in its stoichiometry and interface structure, in which one AMAP1 proline-rich peptide binds to two cortactin SH3 domains simultaneously. We made a cell-permeable peptide derived from the AMAP1 peptide, and we show that this peptide specifically blocks AMAP1/cortactin binding, but not other canonical SH3/proline bindings, and effectively inhibits breast cancer invasion and metastasis. Moreover, this peptide was found to block invasion of other types of cancers, such as glioblastomas and lung carcinomas. We also found that a small-molecule compound, UCS15A, which was previously judged as a weak inhibitor against canonical SH3/proline bindings, effectively inhibits AMAP1/cortactin binding and breast cancer invasion and metastasis. Together with fine structural analysis, we propose that the AMAP1/cortactin complex, which is not detected in normal mammary epithelial cells, is an excellent drug target for cancer therapeutics.
• 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.
• Regulation of Bin1 SH3 domain binding by phosphoinositides

  C Kojima, A Hashimoto, Yabuta, I, M Hirose, S Hashimoto, Y Kanaho, H Sumimoto, T Ikegami, H Sabe

  EMBO JOURNAL 23 (22) 4413 - 4422 0261-4189 2004/11 [Refereed][Not invited]
   

  Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5- bisphosphate (PI(4,5) P-2), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5) P-2 in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5) P-2-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein - protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.
• A novel mode of action of an ArfGAP, AMAP2/PAG3/Pap alpha, in Arf6 function

  S Hashimoto, A Hashimoto, A Yamada, C Kojima, H Yamamoto, T Tsutsumi, M Higashi, A Mizoguchi, R Yagi, H Sabe

  JOURNAL OF BIOLOGICAL CHEMISTRY 279 (36) 37677 - 37684 0021-9258 2004/09 [Refereed][Not invited]
   

  Previously we reported that AMAP2/PAG3/Papalpha/ KIAA0400, a GTPase-activating protein ( GAP), acts to antagonize Arf6 function when overexpressed, whereas it was shown to exhibit efficient GAP activities for other Arf isoforms in vitro. Here, we found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6 but not to GDP-Arf6 or other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function; it exhibits efficient catalytic GAP activity for the class I and II Arfs and yet is involved in the cellular function of the class III Arf without immediate GAP activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.
• 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.
• Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-gamma 2 to Ras in B cell receptor signaling

  M Oh-Hora, S Johmura, A Hashimoto, M Hikida, T Kurosaki

  JOURNAL OF EXPERIMENTAL MEDICINE 198 (12) 1841 - 1851 0022-1007 2003/12 [Refereed][Not invited]
   

  Two important Ras guanine nucleotide exchange factors, Son of sevenless (Sos) and Ras guanine nucleotide releasing protein (RasGRP), have been implicated in controlling Ras activation when cell surface receptors are stimulated. To address the specificity or redundancy of these exchange factors, we have generated Sos1/Sos2 double- or RasGRP3-deficient B cell lines and determined their ability to mediate Ras activation upon B cell receptor (BCR) stimulation. The BCR requires RasGRP3; in contrast, epidermal growth factor receptor is dependent on Sos1 and Sos2. Furthermore, we show that BCR-induced recruitment of RasGRP3 to the membrane and the subsequent Ras activation are significantly attenuated in phospholipase C-gamma2-deficient B cells. This defective Ras activation is suppressed by the expression of RasGRP3 as a mernbrane-attached form, suggesting that phospholipase C-gamma2 regulates RasGkP3 localization and thereby Ras activation.
• Coupling between B cell receptor and phospholipase C-gamma 2 is essential for mature B cell development

  M Hikida, S Johmura, A Hashimoto, M Takezaki, T Kurosaki

  JOURNAL OF EXPERIMENTAL MEDICINE 198 (4) 581 - 589 0022-1007 2003/08 [Refereed][Not invited]
   

  Two signaling pathways known to be essential for progression from miniature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C (PLC)-gamma2 is required for a BAFF-R-mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-gamma2(-/-) mice is caused by a defect in either BCP, or BAFF-R signaling. We find that a PLC-gamma2 SH2 mutant, which inhibits coupling between BCR and PLC-gamma2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R-mediated survival signal, provided by PLC-gamma2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-gamma2 by BCR is required for B cell development.
• Interaction of paxillin with p21-activated kinase (PAK) - Association of paxillin alpha with the kinase-inactive and the Cdc42-activated forms of PAK3

  S Hashimoto, A Tsubouchi, Y Mazaki, H Sabe

  JOURNAL OF BIOLOGICAL CHEMISTRY 276 (8) 6037 - 6045 0021-9258 2001/02 [Not refereed][Not invited]
   

  p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. We show here that paxillin can bind directly to PAK3. We examined several representative focal adhesion proteins, and found that paxillin is the sole protein that associates with PAK3. PAK3 associated with the alpha and beta isoforms of paxillin, but not with gamma. We also show that paxillin alpha associated with both the kinase-inactive and the Cdc42-activated forms of PAKS in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger, beta PIX. Our results revealed that paxillin a can compete with Nck and beta PIX in the binding of PAKS. Moreover, paxillin alpha can be phosphorylated by PAKS at serine. Therefore, paxillin alpha, but not gamma, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and beta PIX, as Nck links PAK1 to growth factor receptors. Our results also revealed that paxillin is involved in highly complexed protein-protein interactions in integrin signaling.
• Paxillin alpha and p130Cas exert opposing effects on cell migration and contact inhibition of growth through their tyrosine phosphorylation.

  H Yano, H Uchida, T Iwasaki, M Mukai, H Akedo, A Tsubouchi, K Nakamura, S Hashimoto, H Sabe

  MOLECULAR BIOLOGY OF THE CELL 11 179A - 180A 1059-1524 2000/12 [Not refereed][Not invited]
  
• A new paxillin-binding protein, PAG3/Pap alpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration

  A Kondo, S Hashimoto, H Yano, K Nagayama, Y Mazaki, H Sabe

  MOLECULAR BIOLOGY OF THE CELL American Society for Cell Biology ({ASCB}) 11 (4) 1315 - 1327 1059-1524 2000/04 [Refereed][Not invited]
   

  Paxillin acts as an adaptor molecule in integrin signaling. Paxillin is localized to focal contacts but seems to also exist in a relatively large cytoplasmic pool. Here, we report the identification of a new paxillin-binding protein, FAGS (paxillin-associated protein with ADP-ribosylation factor [ARF] GTPase-activating protein [GAP] activity, number 3), which is involved in regulation of the subcellular localization of paxillin. FAGS bound to all paxillin isoforms and was induced during monocyte maturation, at which time paxillin expression is also increased and integrins are activated. PAG3 was diffusely distributed in the cytoplasm in premature monocytes but became localized at cell periphery in mature monocytes, a fraction of which then colocalized with paxillin. PAG3, on the other hand, did not accumulate at focal adhesion plaques, suggesting that PAG3 is not an integrin assembly protein. PAG3 was identical to KIAA0400/Pap alpha, which was previously identified as a Pyk2-binding protein bearing a GAP activity toward several ARFs in vitro. Mammalian ARFs fall into three classes, and we showed that all classes could affect subcellular localization of paxillin. We also examined possible interaction of FAGS with ARFs and showed evidence that at least one of them, ARF6, seems to be an intracellular substrate for GAP activity of FAGS. Moreover, overexpression of PAG3, but not its GAP-inactive mutant, inhibited paxillin recruitment to focal contacts and hampered cell migratory activities, whereas cell adhesion activities were almost unaffected. Therefore, our results demonstrate that paxillin recruitment to focal adhesions is not mediated by simple cytoplasmic diffusion; rather, PAG3 appears to be involved in this process, possibly through its GAP activity toward ARF proteins. Our result thus delineates a new aspect of regulation of cell migratory activities.
• Inhibitory modulation of B cell receptor-mediated Ca2+ mobilization by Src homology 2 domain-containing inositol 5 '-phosphatase (SHIP)

  A Hashimoto, K Hirose, H Okada, T Kurosaki, M Iino

  JOURNAL OF BIOLOGICAL CHEMISTRY 274 (16) 11203 - 11208 0021-9258 1999/04 [Refereed][Not invited]
   

  Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) mediates inhibitory signals that attenuate intracellular Ca2+ mobilization in B cells upon B cell receptor (BCR) stimulation. To clarify the mechanisms affected by SHIP, we analyzed Ca2+ mobilization in the DT40 B cell line in which the SHIP gene was disrupted. In SHIP-deficient cells, Ca2+ transient elicited by BCR stimulation was more prolonged than that in control cells both in the presence and absence of extracellular Ca2+, Inositol 1,4,5-trisphosphate production following BCR stimulation was enhanced in SHIP-deficient cells. In SHIP-deficient cells in comparison with the control cells, BCR stimulation in the absence of extracellular Ca2+ induced a greater degree of Ca2+ store depletion and the Ca2+ influx upon re-addition of extracellular Ca2+ was also greater. However, store-operated Ca2+ influx (SOC) elicited by thapsigargin-induced store depletion was not affected by SHIP. These results indicate that the primary target pathway of SHIP is the Ca2+ release from the stores, and that Ca2+ influx by the SOC mechanism is secondarily controlled by the level of Ca2+ in the stores without direct inhibition of SOC. In this way, SHIP may play an important role in ensuring the robust tuning of Ca2+ signaling in B cells.
• Mitosis specific serine phosphorylation and downregulation of one of the focal adhesion protein, paxillin

  R Yamaguchi, Y Mazaki, K Hirota, S Hashimoto, H Sabe

  ONCOGENE 15 (15) 1753 - 1761 0950-9232 1997/10 [Refereed][Not invited]
   

  Mitotic cells typically lack well-formed focal adhesions, As an approach to explore the dynamic process regulating the focal adhesion assembly, we examined states of focal adhesion proteins during mitosis of the cell cycle. We found that the amount of paxillin was significantly reduced during mitosis of the cell cycle, whereas other focal adhesion proteins including talin, vinculin and Focal Adhesion Kinase did not. Proteolytic degradation appeared to be involved in the mitotic reduction, but transcriptional and/or translational controls of the mRNA were not essential for this downregulation. Moreover, concurrent with the decreased protein level, phosphorylation status of paxillin altered during mitosis; mitotic paxillin was phosphorylated primarily on serine and dephosphorylated on tyrosine while interphase one was phosphorylated both on serine and tyrosine. We found that mitotic phosphorylation created an electrophoretically slow-migrating population of paxillin which was barely detected in interphase cells. This mitotic specific modification occurred with both alpha and beta isoforms of paxillin. We also examined the fate of paxillin protein by changing its protein amount. We found that majority of paxillin overexpressed was subjected to the specific modification but not to the downregulation in the mitotic arrested cells. On the other hand, paxillin exogenously expressed at a moderate level was subjected to both the mitotic modification and downregulation. Collectively, we concluded that paxillin's specific serine phosphorylation together with the proteolytic downregulation of a limited fraction of paxillin is taken place during the mitosis of the cell cycle.

MISC

• 癌細胞ARF6-AMAP1経路阻害は抗PD-1抗体と協調的に作用する

  橋本あり, 半田悠, 畑宗一郎, 蔦保暁生, 蔦保暁生, 吉田隆雄, 平野聡, 橋本茂, 橋本茂, 佐邊壽孝  日本生化学会大会(Web)  94th-  2021
• High expression of AMAP1, an ARF6 effector, relates to elevated levels of PD-L1 and fibrosis of pancreatic cancer

  橋本あり, 蔦保暁生, 蔦保暁生, 橋本茂, 橋本茂, 畑宗一郎, 加地紫苑, 平野聡, 佐邊壽孝  日本癌学会学術総会抄録集(Web)  79th-  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
• 膵臓癌のPD-L1発現と線維化におけるARF6-AMAP1経路の役割

  蔦保暁生, 橋本あり, 橋本茂, 佐邊壽孝, 平野聡  日本外科学会定期学術集会(Web)  120th-  2020
• 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]
  
• 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]
  
• 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  [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]
  
• 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)  -2  2016  [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(Web)  89th-  ROMBUNNO.1P‐268 (WEB ONLY)  -268]  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]
  
• 腎盂尿管癌におけるEPB4.1L5発現の意義

  大門 達明, 小坂 威雄, 菊地 栄次, 三上 修治, 宮崎 保匡, 橋本 あり, 橋本 茂, 水野 隆一, 宮嶋 哲, 岡田 保典, 佐邊 壽孝, 大家 基嗣  日本癌学会総会記事  74回-  P  -3251  2015/10  [Not refereed][Not invited]
  
• 腎盂尿管癌におけるEPB4.1L5発現の意義

  大門 達明, 小坂 威雄, 菊地 栄次, 三上 修治, 宮崎 保匡, 橋本 あり, 橋本 茂, 水野 隆一, 宮嶋 哲, 岡田 保典, 佐邊 壽孝, 大家 基嗣  日本癌学会総会記事  74回-  P  -3251  2015/10  [Not refereed][Not invited]
  
• EZH2発現亢進により創出されるArf6を中心とした間葉浸潤に特化した分子装置は腎癌の予後不良に関与する(EZH2 generates Arf6-based mesenchymal invasion machinery that is central to poor prognosis of renal cancer)

  橋本 茂, 杉野 弘和, 橋本 あり, 吉河 歩, 及川 司, 半田 悠, 大家 基嗣, 三上 修治, 佐邊 壽孝  日本癌学会総会記事  73回-  J  -2075  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]
  
• ArfGAPs: Not only for the termination

  Shigeru Hashimoto, Ari Hashimoto, Hirokazu Sugino, Ayumu Yoshikawa, Haruka Handa, Masanao Yoshino, Yutaro Otsuka, Hisataka Sabe  Ras Superfamily Small G Proteins: Biology and Mechanisms 2: Transport  253  -274  2014/05/01  [Not refereed][Not invited]
   

  © 2014 Springer International Publishing Switzerland. All rights reserved.While Arf-family small GTPases (Arf-GTPases) consist of 5 members in humans, 31 human genes have been identified that encode proteins bearing the GTPase-activating protein (GAP) domain for Arf-GTPases. Interestingly, Arf1, the first identified Arf, was shown to substantially lack intrinsic GTPase activity, which other Ras-superfamily members of small GTPases generally bear. Likewise, ArfGAP domains primarily consist of zinc-finger structures, and do not resemble GAP domains for other small GTPases. Arfs primarily function in intracellular vesicle/membrane trafficking. A general model shows that Arfs play roles in membrane budding, in which GTP-Arfs recruit coatomer proteins to generate and maintain membrane curvature to initiate the budding. Coatomers are thought to be separated from Arf-mediated vesicles before they reach the target membrane, while this separation may or may not be coupled with the GTP hydrolysis activity. We have shown that several ArfGAPs, such as AMAP1 and AMAP2, have the ability to bind stably to GTP-Arf6, without immediate GTP hydrolysis. They each contain a BAR domain and hence may act as coatomers for Arf-mediated vesicles. These ArfGAPs moreover act to recruit their binding proteins to sites of Arf6 activation, which are not coatomer components. These findings have amended the classical, general model of the functions of ArfGAPs, as well as Arf-GTPases. In this review, we will describe the recent information revealed about ArfGAPs, with the aim to decipher and discuss their fundamental roles.
• 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]
  
• 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(Web)  86th-  2S04A-3 (WEB ONLY)  -3  2013  [Not refereed][Not invited]
  
• Co-overexpression of GEP100 With EGFR Correlates With Early Recurrence After Breast Conservation Therapy (BCT)

  R. Kinoshita, J. Nam, M. Hosoda, C. Kubota K, M. Tanino, A. Hashimoto, Y. M. Ito, S. Tanaka, H. Sabe, H. Shirato  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  84-  (3)  S227  -S228  2012/11  [Not refereed][Not invited]
  
• EZH2によるmiR-203発現抑制が乳癌浸潤に中枢的であるArf6-AMAP1経路創出に関わる(EZH2-mediated downregulation of miR-203 generates the Arf6-AMAP1 pathway pivotal for breast cancer invasiveness)

  杉野 弘和, 橋本 茂, 橋本 あり, 吉河 歩, 半田 悠, 佐邊 壽孝  日本癌学会総会記事  71回-  87  -87  2012/08  [Not refereed][Not invited]
  
• がんの浸潤・転移に関与するGEP100-Arf6-AMAP1経路とc-Metシグナルとの相互作用(GAB1 links c-Met signaling with GEP100-Arf6-AMAP1 pathway to promote breast cancer invasiveness)

  吉河 歩, 橋本 茂, 橋本 あり, 杉野 弘和, 大塚 勇太郎, 味藤 静, 半田 悠, 佐邊 壽孝  日本癌学会総会記事  71回-  87  -87  2012/08  [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, 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-  WEB ONLY 2W10II-1  2012  [Not refereed][Not invited]
  
• Arf6活性化による乳癌浸潤形質獲得過程におけるエピジェネティック制御の関与

  橋本茂, 杉野弘和, 橋本あり, 吉河歩, 大塚勇太郎, 芳野正修, 半田悠, 佐邊壽孝  日本分子生物学会年会プログラム・要旨集(Web)  35th-  WEB ONLY 2P-0166  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]
  
• 過剰発現したHer2/Neu/ErbB2とGEP100/BRAG2の連係は肺腺癌の自律的な浸潤活性を誘導し、転移を予測するバイオマーカーを提供する(Engagement of GEP100/BRAG2 with overexpressed Her2/Neu/ErbB2 induces autonomous invasive activities and provides a biomarker predictive for metastases of lung adenocarcinoma)

  毛受 暁史, 橋本 茂, 橋本 あり, 伊達 洋至, 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  -0238  2010/12  [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]
  
• HER2はGEP100を介して肺癌の浸潤転移を促進する(ErbB2/Her2/Neu employs GEP100 to promote lung cancer invasion and metastasis)

  毛受 暁史, 橋本 茂, 橋本 あり, 伊達 洋至, 佐邊 壽孝  日本癌学会総会記事  69回-  266  -266  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]
  
• GEP100 binding to HER2 receptor promotes lung cancer invasion and metastasis

  Toshi Menju, Shigeru Hashimoto, Ari Hashimoto, Tsuyoshi Takahashi, Ei Nakayama, Ryutaro Kikuchi, Masashi Ishikawa, Masashi Kobayashi, Jiro Kitamura, Makoto Sonobe, Ryo Miyahara, Kenichi Ookubo, Hiroshi Date, Hisataka Sabe  JOURNAL OF THORACIC ONCOLOGY  5-  (6)  S228  -S228  2010/06  [Not refereed][Not invited]
  
• GEP100-Arf6-AMAP1経路は癌浸潤と血管新生に対する共通の分子標的である(GEP100-Arf6-AMAP1 pathway is activated by VEGFR2 and promotes vascular remodeling and VE-cadherin endocytosis in endothelial cells)

  橋本 あり, 橋本 茂, 小川 栄治, 毛受 暁史, 森重 真毅, 佐邊 壽孝  日本細胞生物学会大会講演要旨集  62回-  161  -161  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]
  
• Arf6-AMAP1 pathway in invasion of lung cancer and malignant mesothelioma cell lines

  T. Menju, S. Hashimoto, A. Hashimoto, E. Ogawa, H. Wada, H. Date, H. Sabe  EJC SUPPLEMENTS  6-  (12)  55  -55  2008/10  [Not refereed][Not invited]
  
• The EGFR-GEP100-Arf6 pathway in breast cancer invasion and metastasis

  S. Hashimoto, M. Morishige, E. Ogawa, Y. Toda, H. Kotani, M. Hirose, A. Hashimoto, Y. Nio, H. Wada, H. Sabe  EJC SUPPLEMENTS  6-  (12)  53  -54  2008/10  [Not refereed][Not invited]
  
• Common usage of the GEP100-Arf6-AMAP1 pathway in tumor invasion, angiogenesis and vascular permeability

  A. Hashimoto, S. Hashimoto, E. Ogawa, M. Hirose, M. Morishige, T. Menju, M. Shibuya, H. Sabe  EJC SUPPLEMENTS  6-  (12)  20  -20  2008/10  [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]
  
• 低酸素環境とEMTにおける乳腺上皮細胞の浸潤性獲得に関連する網羅的解析(Comprehensive analysis for the acquisition of invasiveness of mammary epithelial cells under hypoxia and EMT)

  橋本 茂, 橋本 あり, 魏 樹梅, 三浦 浩一, 毛受 暁史, 佐邊 壽孝  日本癌学会総会記事  67回-  101  -101  2008/09  [Not refereed][Not invited]
  
• GEP100-Arf6経路は血管新生と癌浸潤に共通のシグナル経路である(Common usage of the GEP100-Arf6 signaling pathway in tumor invasion, angiogenesis, and vascular permeability)

  橋本 あり, 橋本 茂, 小川 栄治, 廣瀬 まゆみ, 森重 真毅, 毛受 暁史, 渋谷 正史, 佐邊 壽孝  日本癌学会総会記事  67回-  296  -296  2008/09  [Not refereed][Not invited]
  
• 癌浸潤転移における細胞運動のメカニズム 血管新生と癌浸潤に共通なシグナル経路(Molecular mechanisms of cell migration in cancer invasion and metastasis Common usage of an Arf6-GEP100 signaling pathway in angiogenesis and tumor invasion)

  橋本 あり, 橋本 茂, 小川 栄治, 廣瀬 まゆみ, 高島 成二, 森重 真毅, 毛受 暁史, 南 ジンミン, 真崎 雄一, 北風 政史, 渋谷 正史, 佐邊 壽孝  日本細胞生物学会大会講演要旨集  60回-  95  -95  2008/06  [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]
  
• EMTと低酸素下での浸潤活性獲得に共通なエピジェネティック変化

  橋本茂, 三浦浩一, 橋本あり, WEI Shumei, 毛受暁史, 毛受暁史, 佐邊壽孝  生化学  2P-0426  2008  [Not refereed][Not invited]
  
• EGFレセプターシグナルを介した乳癌細胞の浸潤形質誘導における必須因子Arf6の活性化機序(GEP100 links epidermal growth factor receptor signaling to Arf6 activation to induce breast cancer invasion)

  橋本 茂, 森重 真毅, 小川 栄治, 廣瀬 まゆみ, 魏 樹梅, 橋本 あり, 山田 敦子, 矢野 元, 仁尾 義則, 和田 洋巳, 古林 秀則, 佐邊 壽孝  日本癌学会総会記事  66回-  58  -58  2007/08  [Not refereed][Not invited]
  
• 癌浸潤及び血管新生におけるArf6活性化の意義(Common usage of Arf6-signaling pathway in tumor invasion and angiogenesis)

  橋本 あり, 南 ジンミン, 森重 真毅, 毛受 暁史, 橋本 茂, 渋谷 正史, 佐邊 壽孝  日本癌学会総会記事  66回-  142  -142  2007/08  [Not refereed][Not invited]
  
• 乳癌細胞の浸潤形質獲得における必須因子Arf6の活性化機序

  橋本茂, 森重真毅, 森重真毅, 古林秀則, 橋本あり, WEI Shumei, 山田敦子, 佐邊壽孝  日本癌学会学術総会記事  65th-  456  2006/08/28  [Not refereed][Not invited]
  
• AMAP1/コータクチン複合体形成阻害による血管新生及びmesenchymal型とamoeboid型癌浸潤の阻害

  橋本あり, 山田敦子, 森重真毅, 森重真毅, 橋本茂, 佐邊壽孝, 佐邊壽孝  日本癌学会学術総会記事  65th-  311  2006/08/28  [Not refereed][Not invited]
  
• 癌細胞浸潤阻害剤の標的に成り得るAMAP1/コータクチンのインタフェイスの構造

  廣瀬まゆみ, 廣瀬まゆみ, 橋本茂, 橋本あり, 森重真毅, 森重真毅, 山田敦子, 保坂晴美, 赤木謙一, 小川栄治, 小川栄治, 池上貴久, 中川敦史, 佐邊壽孝  日本蛋白質科学会年会プログラム・要旨集  6th-  67  2006/03/31  [Not refereed][Not invited]
  
• EMT-inducible Protein, Erythrocyte Protein Band4.1 Like-5, Plays an Essential Role for the EMT Progression

  M. Hirano, S. Hashimoto, H. Sabe, S. Aizawa  MOLECULAR BIOLOGY OF THE CELL  17-  2006  [Not refereed][Not invited]
  
• 乳癌細胞と単球細胞におけるAMAP1蛋白質量の異なる調節機構

  宮田真理子, 宮田真理子, 橋本茂, 橋本あり, 佐辺寿孝, 佐辺寿孝  日本分子生物学会年会講演要旨集  28th-  459  2005/11/25  [Not refereed][Not invited]
  
• 好中球化学走行において細胞極性と活性酸素産生方向を繋ぐ仕組みについて

  真崎雄一, 橋本あり, 森重真毅, 森重真毅, NAM Jin‐Min, 橋本茂, 佐辺寿孝, 佐辺寿孝  日本分子生物学会年会講演要旨集  28th-  81  2005/11/25  [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, 広瀬まゆみ, 橋本あり, 森重真毅, 森重真毅, 山田敦子, 小野寺康仁, 小野寺康仁, 小川栄治, 和田洋巳, 池上貴久, 中川敦史, 佐辺寿孝, 佐辺寿孝  日本癌学会学術総会記事  64th-  309  2005/08/15  [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

  佐辺寿孝, 小野寺康仁, 小野寺康仁, NAM Jinmin, 橋本あり, 森重真毅, 森重真毅, 橋本茂  日本癌学会学術総会記事  64th-  513  2005/08/15  [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, 佐辺寿孝  日本癌学会総会記事  63rd-  265  2004/08/25  [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, 佐辺寿孝  日本癌学会総会記事  63rd-  267  2004/08/25  [Not refereed][Not invited]
  
• A novel mode of action of an ArfGAP, AMAP2/PAG3/Papa, in Arf6 function

  Shigeru Hashimoto, Ari Hashimoto, Atsuko Yamada, Hiroko Yamamoto, Chie Kojima, Tomonari Tsutsumi, Mikito Higashi, Akira Mizoguchi, Ryohei Yagi, Hisataka Sabe  CELL STRUCTURE AND FUNCTION  29-  109  -109  2004/05  [Not refereed][Not invited]
  
• PAG3/Pap alpha/KIAA0400, an ARFGAP, interacts with GTP-bound ARF6 and endocytic proteins

  S Hashimoto, A Hashimoto, A Yamada, C Kojima, H Yamamoto, A Kondo, R Yagi, H Sabe  MOLECULAR BIOLOGY OF THE CELL  13-  473A  -474A  2002/11  [Not refereed][Not invited]
  
• 抗原受容体を介するB細胞分化制御の分子機構の解析

  疋田正喜, 上村幸子, 橋本あり, 黒崎知博  日本免疫学会総会・学術集会記録  32-  93  2002/10/31  [Not refereed][Not invited]
  
• PLC‐γ2を介するB細胞分化の分子機序の解析

  疋田正喜, 上村幸子, 橋本あり, 黒崎知博  生化学  74-  (8)  685  2002/08/25  [Not refereed][Not invited]
  
• パキシリン結合性ARF6GAP(PAG3)による細胞運動性制御の解析

  橋本あり, 山本裕子, 八木良平, 近藤明子, 橋本茂, 佐辺寿孝  生化学  73-  (8)  756  2001/08/25  [Not refereed][Not invited]
  
• An ADP-ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is involved in subcellular localization of paxillin and actin cytoskeletal organization

  Y Mazaki, S Hashimoto, K Okawa, A Tsubouchi, K Nakamura, R Yagi, H Yano, A Kondo, A Iwamatsu, A Mizoguchi, H Sabe  MOLECULAR BIOLOGY OF THE CELL  12-  (3)  645  -662  2001/03  [Not refereed][Not invited]
   

  Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP? activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAF-inactive mutant, caused the redistribution of Golgi protein beta -COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.
• Interaction of paxillin with p21-activated kinase (PAK)

  S Hashimoto, A Tsubouchi, Y Mazaki, H Sabe  MOLECULAR BIOLOGY OF THE CELL  11-  172A  -173A  2000/12  [Not refereed][Not invited]
  
• PLC‐γ2欠損マウスの解析

  橋本あり, 竹田潔, 稲葉宗夫, 関亦正幸, 改正恒康, 池原進, 本間好, 審良静男, 黒崎知博  日本免疫学会総会・学術集会記録  30-  331  2000/09/26  [Not refereed][Not invited]
  
• Cutting edge: Essential role of phospholipase C-gamma 2 in B cell development and function

  A Hashimoto, K Takeda, M Inaba, M Sekimata, T Kaisho, S Ikehara, Y Homma, S Akira, T Kurosaki  JOURNAL OF IMMUNOLOGY  165-  (4)  1738  -1742  2000/08  [Not refereed][Not invited]
   

  Cross-linking of the B cell Ag receptor (BCR) induces the tyrosine phosphorylation of multiple cellular substrates, including phospholipase C (PLC)-gamma 2, which is involved in the activation of the phosphatidylinositol pathway. To assess the importance of PLC-gamma 2 in murine lymphopoiesis, the PLC-gamma 2 gene was inducibly ablated by using IFN-regulated Cre recombinase. Mice with a neonatally induced loss of PLC-gamma 2 function displayed reduced numbers of mature conventional B cells and peritoneal B1 cells and defective responses in vitro to BCR stimulation and in vivo to immunization with thymus-independent type II Ags. In contrast, T cell development and TCR-mediated proliferation were normal. Taken together, PLC-gamma 2 is a critical component of BCR signaling pathways and is required to promote B cell development.
• HPK1 is activated by lymphocyte antigen receptors and negatively regulates AP-1

  J Liou, F Kiefer, A Dang, A Hashimoto, MH Cobb, T Kurosaki, A Weiss  FASEB JOURNAL  14-  (6)  A1177  -A1177  2000/04  [Not refereed][Not invited]
  
• Regulation of the phospholipase C-γ2 pathway in B cells

  T. Kurosaki, A. Maeda, M. Ishiai, A. Hashimoto, K. Inabe, M. Takata  Immunological Reviews  176-  19  -29  2000  [Not refereed][Not invited]
   

  In B lymphocytes, a signaling complex that contributes to cell fate decisions is the B-cell antigen receptor (BCR), with different extents of receptor engagement leading to such outcomes as cell death, survival, or proliferation. Here, based upon the available genetic and biochemical data of the BCR signal components, we discuss several mechanisms by which BCR signals are propagated and modified, with specific emphasis on the phospholipase C (PLC)-γ2-calcium pathway. Gene-targeting experiments in DT40 chicken B cells highlighted the importance of the intracellular protein tyrosine kinases Syk and Btk in PLC-γ2 activation. Until recently, the molecular mechanism underlying the double requirement for Syk and Btk in PLC-γ2 activation remained unclear, but new data suggest that an adapter molecule, B-cell linker protein (alternatively named SLP-65 or BASH), phosphorylated by Syk, provides docking sites for Btk SH2 domain as well as PLC-γ2 SH2 domains, thus bringing Btk into close proximity with PLC-γ2. The activated Btk then phosphorylates PLC-γ2, leading to its activation. The activated PLC-γ2 converts phosphatidylinositol 4,5-bisphosphate into the second messenger inositol 1,4,5-trisphosphate (IP3), which in turn binds to IP3 receptors located on the endoplasmic reticulum (ER). Binding of IP3 to the IP3 receptors is essential for triggering a calcium release from the ER and subsequent entry of extracellular calcium. Balancing these activation signals in the PLC-γ2-calcium pathway are the inhibitory receptors expressed on B cells, FcγRII and paired immunoglobin-like receptor (PIR)-B. Although both FcγRII and PIR-B inhibits the BCR-mediated [Ca2+](i) increase, the inhibitory mechanisms of these receptors are distinct. The FcγRII-mediated inhibitory signal is dependent on lipid phosphatase SHIP, whereas the PIR-B requires redundant functions of protein phosphatases SHP-1 and SHP-2. Thus, PIR-B and FcγRII inhibit calcium signals by utilizing two distinct signaling molecules, thereby contributing to setting threshold levels for activation signals as well as terminating activation responses.
• EGF刺激によるERK,JNK活性化シグナルにおけるGrb2及びShcの機能解析

  橋本あり, 後藤典子, 渋谷正史, 黒崎知博  日本癌学会総会記事  58th-  663  1999/08/30  [Not refereed][Not invited]
  
• Shc regulates epidermal growth factor-induced activation of the JNK signaling pathway

  A Hashimoto, M Kurosaki, N Gotoh, R Shibuya, T Kurosaki  JOURNAL OF BIOLOGICAL CHEMISTRY  274-  (29)  20139  -20143  1999/07  [Not refereed][Not invited]
   

  Two adaptor molecules, Grb2 and Shc, have been implicated in the extracellular signal-regulated kinase (ERK) activation by receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). Here we show that the EGF-mediated ERK activation is abolished by loss of Grb2, whereas this response is not affected by loss of Shc. Conversely, the EGF-mediated c-Jun N-terminal kinase (JNK) activation is dependent on Shc, but not Grb2. These findings strongly support distinct roles for Grb2 and Shc in controlling ERK and JNK activation after EGF stimulation.
• Molecular mechanisms of ERK,JNK and p38 activation upon B cell receptor engagement

  FASEB Conferences にて発表  1999  [Not refereed][Not invited]
  
• Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

  T Suzuki, T Kurosaki, K Shimada, N Kansaku, U Kuhnlein, D Zadworny, K Agata, A Hashimoto, M Koide, M Koike, M Takata, A Kuroiwa, S Minai, T Namikawa, Y Matsuda  CYTOGENETICS AND CELL GENETICS  87-  (1-2)  32  -40  1999  [Not refereed][Not invited]
   

  Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q). Copyright (C) 1999 S. Karger AG, Basel.
• Molecular mechanisms of ERK, JNK and p38 activation upon B cell receptor engagement

  HASHIMOTO Ari, OKADA Hidetaka, JIANG Aimin, KUROSAKI Mari, GREENBERG Steven, A. CLARK Edward, KUROSAKI Tomohiro  日本分子生物学会年会プログラム・講演要旨集  21-  (0)  535  -535  1998/12/01  [Not refereed][Not invited]
  
• B細胞レセプターを介するERK,JNK,p38の活性化分子機構

  橋本あり, 岡田英孝, JIANG A, 黒崎まり, GREENBERG S, CLARK E A, 黒崎知博  日本分子生物学会年会プログラム・講演要旨集  21st-  535  1998/11  [Not refereed][Not invited]
  
• Involvement of guanosine triphosphatases and phospholipase C-gamma 2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase activation by the B cell antigen receptor

  A Hashimoto, H Okada, A Jiang, M Kurosaki, S Greenberg, EA Clark, T Kurosaki  JOURNAL OF EXPERIMENTAL MEDICINE  188-  (7)  1287  -1295  1998/10  [Not refereed][Not invited]
   

  Mitogen-activated protein (MAP) kinase family members, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase, have been implicated in coupling the B cell antigen receptor (BCR) to transcriptional responses. However, the mechanisms that lead to the activation of these MAP kinase family members have been poorly elucidated. Here we demonstrate that the BCR-induced ERK activation is reduced by loss of Grb2 or expression of a dominant-negative form of Ras, RasN17, whereas this response is not affected by loss of Shc. The inhibition of the ERK response was also observed in phospholipase C (PLC)-gamma 2-deficient DT40 B cells, and expression of RasN17 in the PLC-gamma 2-deficient cells completely abrogated the ERK activation. The PLC-gamma 2 dependency of ERK activation was most likely due to protein kinase C (PKC) activation rather than calcium mobilization, since loss of inositol 1,4,5-trisphosphate receptors did not affect ERK activation. Similar to cooperation of Ras with PKC activation in ERK response, both PLC-gamma 2-dependent signal and GTPase are required for BCR-induced JNK and p38 responses. JNK response is dependent on Rac1 and calcium mobilization, whereas p38 response requires Rad and PKC activation.
• Degradation of decorin by matrix metalloproteinases: Identification of the cleavage sites, kinetic analyses and transforming growth factor-beta 1 release

  K Imai, A Hiramatsu, D Fukushima, MD Pierschbacher, Y Okada  BIOCHEMICAL JOURNAL  322-  809  -814  1997/03  [Not refereed][Not invited]
   

  Decorin (DCN) is a ubiquitous proteoglycan comprised of a core protein attached to a single dermatan/chondroitin sulphate glycosaminoglycan chain. It may play a role in regulation of collagen fibrillogenesis and function as a reservoir of transforming growth factor beta (TGF-beta) in the extracellular milieu. We have examined the susceptibility of DCN to five different matrix metalloproteinases (MMPs): MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin) and MMP-9 (gelatinase B). MMP-2 and MMP-3 digest DCN into seven major fragments in a similar pattern. The N-terminal sequence of the two fragments generated by MMP-2 and MMP-3 is Leu(211)-Lys-Gly-Leu-Asn, but that of the others is Asp(1)-Glu-Ala-Ser-Gly. MMP-7 cleaves DCN into three major fragments which have the N-termini Asp(1)-Glu-Ala-Ser-Gly, Glu(2)-Ala-Ser-Gly-Ile and Leu(244)-His-Leu-Asp-Asn. Activities of MMP-1 and MMP-9 against DCN are negligible. The values of K-m for the MMPs capable of degrading DCN are very similar (10-12 mu M), but the k(eat)/K-m value for MMP-7 (30.5 mu M(-1).(-1)) is 4.5-fold higher than those for MMP-2 and MMP-3. Incubation of a DCN-TGF-beta 1 complex with MMP-2, -3 or -7 results in release of TGF-beta 1 from the complex. These data indicate proteolytic degradation of DCN by MMP-2, MMP-3 and MMP-7, and suggest the possibility that, under pathophysiological conditions, the digestion by the MMPs may induce tissue reactions mediated by TGF-beta 1 released from DCN in the connective tissues.
• プロテインキナーゼC-C2ドメインの大量調製とカルシウム結合反応

  日本生物物理学会発表  1993  [Not refereed][Not invited]
  
• バキュロウイルス-昆虫細胞系を用いたプロテインキナーゼC構造ドメインの発現

  修士学位論文  1993  [Not refereed][Not invited]
  
• Large-scale preparotion of the C2-domain of protein kinase C and its interaction with calcium.

  1993  [Not refereed][Not invited]
  
• Expression of the functional Protein Kinase C domains using basulovirus-infected cells : purification and structual analysis.

  1993  [Not refereed][Not invited]
  

Association Memberships

• JAPAN SOCIETY FOR CELL BIOLOGY   日本生化学会   日本癌学会   日本分子生物学会   

Research Projects

• ARF6-AMAP1経路による細胞内動態変動と免疫回避作動の分子機序の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2022/04 -2025/03 

  Author : 橋本 あり
• Regulatory mechanisms of immune checkpoint PD-L1 via the Arf6 pathway in progressive cancer

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

  Date (from‐to) : 2017/04 -2022/03 

  Author : Hashimoto Ari

   

  We previously showed that the small GTPase Arf6 and its downstream effector AMAP1 constitute the core signaling machinery that drives cancer malignancy and therapeutic resistance in breast and renal cancers. In this project, we showed that mutations in KRAS/TP53, the major driver oncogenes of pancreatic cancer, cooperatively cause the overexpression of Arf6 and AMAP1 proteins and activation of the Arf6-AMAP1 pathway to promote PDAC invasion, metastasis, fibrosis, and immune evasion. Mechanistically, the Arf6-AMAP1 pathway promoted recycling of the immune checkpoint molecule PD-L1 to the cell membrane surface. We also found that the epigenetic factor EZH2 is involved in the regulation of Arf6 and PD-L1 expression, and identified candidate molecules for its regulation. Moreover, we demonstrated that inhibition of the Arf6-AMAP1 pathway in cancer cells results in therapeutic synergy with an anti-PD-1 antibody, and is hence a novel method for improving the efficacy of anti-PD-1 therapies.
• Elucidation of the molecular mechanisms of early breast cancer invasion and metastasis

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

  Date (from‐to) : 2011 -2013 

  Author : HASHIMOTO Ari

   

  Cancer progression by which epithelial tumor cells acquire a more motile and invasive phenotype has been closely linked to the epithelial-mesenchymal transition (EMT) process. We found that GEP100-Arf6-AMAP1 pathway is important for acquisition of mesenchymal invasive phenotypes of breast cancers and TP53 alterations link to this process. Metabolic pathway regulated by mutant p53 is required for Arf6 activation. The clinical specimens of human breast cancer showed that the expression of GEP100-Arf6-AMAP1 signal correlated with recurrence after radiotherapy. Database analysis also showed that co-incidence of TP53 mutations and the high expression of this signal pathway statistically correlate with poor survival. These results suggest that generation of the Arf6-based mesenchymal invasive pathway by TP53 alterations regulate malignant progression and appears to be crucial for poor prognosis in breast cancer.
• 腫瘍血管における透過性亢進に関与する分子輸送のメカニズム及び病態の解明

  文部科学省：科学研究費補助金(新学術領域研究(研究領域提案型))

  Date (from‐to) : 2009 -2010 

  Author : 橋本 あり

   

  血管新生は癌の悪性度進行の律速段階の一つと考えられている。疾患における血管新生の分子機序を明らかにすることが病態の理解と効果的な治療に繋がるものと思われる。腫瘍血管は、脆弱で透過性が亢進しているため、癌転移が生じやすく、薬剤到達が不利と考えられている。血管透過性はVEGF(vascular endothelial growth factor)によって誘導されるVE-cadherinの細胞内輸送の制御が重要な役割を担っている。しかしながら、進行癌における血管透過性の亢進とVE-cadherinの細胞内動態との関連には不明な点が多い。本研究期間では、以下の研究成果を得た。1.GEP100-Arf6-AMAP1シグナルとVE-cadherinとの相互作用に関与する分子の同定上皮細胞の浸潤形質獲得過程のモデルであるEMT(epithelial-mesenchymal transition)において、接着分子E-cadherinの裏打ち蛋白質p120cateninと結合し、E-cadherinのエンドサイトーシスを促進し、細胞間接着の消失に関与する分子EPB41L5は、VEGF刺激後VE-cadherinと共局在することが分かった。また、EPB41L5はVE-cadherinとp120cateninの結合を阻害すること、AMAP1とN末を介して結合することを明らかにした。これらの結果は...
• Analysis of common molecular mechanisms and pharmaceutical research in cancer invasion and angiogenesis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2008 -2010 

  Author : Ari HASHIMOTO

   

  We have shown that GEP100 links EGFR signaling to Arf6 activation to induce invasive activities of some breast cancer cells. Our analyses demonstrate that the same GEP100-Arf6-AMAP1 pathway is essential for VEGF-induced angiogenesis activities, and that VEGFR2, via phospho-Tyr951, binds to the PH domain of GEP100 to activate the Arf6-AMAP1 pathway to induce angiogenesis. Moreover, we found the small compound that inhibits the interaction between GEP100 and VEGFR-2. The GEP100-Arf6-AMAP1 pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion...
• 細胞の浸潤性制御機構の解析

  文部科学省：科学研究費補助金(若手研究(B))

  Date (from‐to) : 2005 -2006 

  Author : 橋本 あり

   

  本研究は、細胞運動制御や細胞の組織への浸潤性をもたらす基本的分子機序を明かにすること、また、癌細胞の浸潤転移性などの正常からの逸脱のメカニズムを明かにしていくことを目的としている。申請者は、これまでに低分子量G蛋白質Arf6が乳癌細胞の浸潤活性において重要な役割を担うこと、また、Arf6のエフェクターとしてAMAP1を見いだし、乳癌細胞の浸潤に必須であること、AMAP1の蛋白質発現量が乳癌の浸潤性と正の相関があることを明らかにしてきた。さらに、乳癌の浸潤性と相関してAMAP1を中心とした複合体が形成されることを見出した。この複合体は、AMAP1/コータクチン/パキシリンから成る。正常組織での細胞浸潤は、胎児期の発生・分化過程や創傷治癒、免疫応答、血管新生等で観察される。これまで見出してきた乳癌細胞の浸潤活性に重要な役割を担う分子装置が、正常組織における細胞浸潤においても機能しているかを中心に解析を行った。本研究期間では、以下の研究成果を得た。1.個体発生、特に乳腺発生におけるAMAPIの発現及び機能解析正常組織における各種臓器やマウス胎児におけるAMAP1の発現、及び乳癌細胞の浸潤に重要であるAMAP1/コータクチン/パキシリン複合体をある種の臓器において確認した。特に、乳腺組織を構成している上皮細胞は、成長、妊娠に伴って細胞浸潤を活発に行っていることが知られており、Arf6...
• Roles played by Arf6 in cell migration and invasion

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2004 -2005 

  Author : Hisataka SABE, 真崎 雄一, 橋本 茂, 三浦 浩一, 橋本 あり

   

  Cell migration is a multifactorial process in which a number of distinct events occur simultaneously. The major purpose of this study is to understand basic molecules and mechanisms coordinately regulating cell migration and invasion.Arf6 plays essential roles in recycling of plasma membrane component, as well as both membrane and cytoskeletal remodeling at cell peripheries. We have previously identified several proteins bearing ArfGAP domains as binding proteins to paxillin, an integrin signaling adaptor/scaffolding protein. These ArfGAPs include AMAP1 and AMAP2. We have also shown that AM...
• Cell motility and the backward bulk flow of the plasma membrane components

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2002 -2003 

  Author : SABE Hisataka, YAGI Ryohei, YANO Hajime, HASHIMOTO Shigeru, HASHIMOTO Ari

   

  Previously we reported that AMAP2/PAG3/Papα/KIAAO400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, while it was shown to exhibit efficient GAPing activities for other Arf isoforms in vitro. During this period of the two fiscal years we first found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6, but not to GDP-Arf6 nor other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures, and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin-IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function ; it exhibits efficient catalytic GAP activity for the class I and III Arfs, and yet is involved in the cellular function of the class III Arf without immediate GAPing activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6. During analyzing physiological roles of AMAP1, another paxillin-binding ArfGAP we have previously isolated, we found that this protein is localized to invadopodia of breast cancer cells and plays an essential role for the invasion. Since AMAP1 acts to antagonize Arf6, we also analyzed possible role of Arf6 in cancer invasion, and found that Arf6 is also essential for breast cancer invasion. We have proposed that Arf6, and the intracellular machinery regulating Arf6 during invasion, should be considered as therapeutic targets for the prevention of breast cancer invasion.
• 細胞運動と接触阻止の分子機構の解析

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  Date (from‐to) : 2001 -2003 

  Author : 橋本 あり

   

  「接触阻止」は、細胞が互いの存在を認識し、それまで個々ばらばらに振る舞っていた状態から、統制のとれた細胞社会として振る舞う為に必須な機序である。接触阻止の分子機序を解析するためには、まず接触阻止を引き起こす前過程である細胞運動の分子機序を解析することが重要である。細胞運動は生命にとって基本的要因の一つであり、それらの破掟はがん等の疾患を引き起こす。そこで、本年度は下記の点について解析を行った。 1 パキシリン結合性ArfGAP蛋白質の作用機序の解析 インテグリン裏打ち蛋白質であるパキシリンに結合する分子として見い出したArfGAP蛋白質(PAG2/AMAP1,PAG3/AMAP2)は、低分子量G蛋白質であるArf6に対してGAP活性を示す。これまでに、PAG3/AMAP2はArf6が関わるリサイクリングに重要であることを示してきた。一方、PAG2/AMAP1は乳癌細胞の浸潤活性に重要であることを新たに見い出した。Arf6が癌の浸潤活性に重要あることも、siRNA法などにより新たに見い出している。これまでに、乳癌細胞の浸潤活性に重要と言われているパキシリン、cortactinの両者にPAG2/AMAP1は結合し、これら三者が複合体を形成している場合に高い浸潤活性を示すことを見い出した。そこで、この三者の複合体形成が、いつどのように行われているのかを明らかにすべく、マウスやヒトでの発現レベルを現在解析している。 2 遺伝子破壊によるin vivoでのArfGAP蛋白質の解析 ArfGAP蛋白質は、細胞の運動性・浸潤能との関連性が示唆される。これらのことを個体レベルで証明するために、ArfGAP蛋白質の条件的遺伝子破壊マウスの作製を行っている。現在、キメラマウスが生まれており、目的のマウスが出来次第、どの時期、どの組織の細胞運動や浸潤過程に関わっているかの解析を行う。
• B細胞レセプターを介するERK,JNK,p38の活性化分子機構
• B細胞におけるPLC-σ2の機能解析
• 細胞運動における分子機構の解析
• Molecular mechanisms of ERK,JNK and p38 activation upon B cell
• Functional analysis of PLC-σ2 in B cell
• Molecular mechanisms of cell migration