GGT1 is a SNP eQTL gene involved in STAT3 activation and associated with the development of Post-ERCP pancreatitis
Ryutaro Furukawa, Masaki Kuwatani, Jing-Jing Jiang, Yuki Tanaka, Rie Hasebe, Kaoru Murakami, Kumiko Tanaka, Noriyuki Hirata, Izuru Ohki, Ikuko Takahashi, Takeshi Yamasaki, Yuta Shinohara, Shunichiro Nozawa, Shintaro Hojyo, Shimpei I. Kubota, Shigeru Hashimoto, Satoshi Hirano, Naoya Sakamoto, Masaaki Murakami
Scientific Reports, 14, 1, Springer Science and Business Media LLC, 28 May 2024, [Peer-reviewed]
Scientific journal, Abstract

Post-ERCP pancreatitis (PEP) is an acute pancreatitis caused by endoscopic-retrograde-cholangiopancreatography (ERCP). About 10% of patients develop PEP after ERCP. Here we show that gamma-glutamyltransferase 1 (GGT1)-SNP rs5751901 is an eQTL in pancreatic cells associated with PEP and a positive regulator of the IL-6 amplifier. More PEP patients had the GGT1 SNP rs5751901 risk allele (C) than that of non-PEP patients at Hokkaido University Hospital. Additionally, GGT1 expression and IL-6 amplifier activation were increased in PEP pancreas samples with the risk allele. A mechanistic analysis showed that IL-6-mediated STAT3 nuclear translocation and STAT3 phosphorylation were suppressed in GGT1-deficient cells. Furthermore, GGT1 directly associated with gp130, the signal-transducer of IL-6. Importantly, GGT1-deficiency suppressed inflammation development in a STAT3/NF-κB-dependent disease model. Thus, the risk allele of GGT1-SNP rs5751901 is involved in the pathogenesis of PEP via IL-6 amplifier activation. Therefore, the GGT1-STAT3 axis in pancreas may be a prognosis marker and therapeutic target for PEP.

DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation
Seiichiro Naito, Hiroki Tanaka, Jing-Jing Jiang, Masato Tarumi, Ari Hashimoto, Yuki Tanaka, Kaoru Murakami, Shimpei I. Kubota, Shintaro Hojyo, Shigeru Hashimoto, Masaaki Murakami
Biochemical and Biophysical Research Communications, 703, 149666, 149666, Elsevier BV, Apr. 2024, [Peer-reviewed], [Corresponding author]
Scientific journal

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, (一社)日本癌学会, Sep. 2023
English

Computer model of IL-6-dependent rheumatoid arthritis in F759 mice
Reiji Yamamoto, Satoshi Yamada, Toru Atsumi, Kaoru Murakami, Ari Hashimoto, Seiichiro Naito, Yuki Tanaka, Izuru Ohki, Yuta Shinohara, Norimasa Iwasaki, Akihiko Yoshimura, Jing-Jing Jiang, Daisuke Kamimura, Shintaro Hojyo, Shimpei I Kubota, Shigeru Hashimoto, Masaaki Murakami
International Immunology, 35, 9, 403, 421, Oxford University Press (OUP), 25 May 2023, [Peer-reviewed], [Corresponding author]
Scientific journal, Abstract

The interleukin-6 (IL-6) amplifier, which describes the simultaneous activation of signal transducer and activator of transcription 3 (STAT3) and NF-κb nuclear factor kappa B (NF-κB), in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-κB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NF-κB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 (interleukin 17) signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-κB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and C-C motif chemokine ligand 2 (CCL2), phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of T helper 17 (Th17) cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

GM-CSF Promotes the Survival of Peripheral-Derived Myeloid Cells in the Central Nervous System for Pain-Induced Relapse of Neuroinflammation
Shiina Matsuyama, Reiji Yamamoto, Kaoru Murakami, Nobuhiko Takahashi, Rieko Nishi, Asuka Ishii, Junko Nio-Kobayashi, Nobuya Abe, Kumiko Tanaka, Jing-Jing Jiang, Tadafumi Kawamoto, Toshihiko Iwanaga, Yuta Shinohara, Takeshi Yamasaki, Izuru Ohki, Shintaro Hojyo, Rie Hasebe, Shimpei I Kubota, Noriyuki Hirata, Daisuke Kamimura, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami
The Journal of Immunology, 211, 1, 34, 42, Oxford University Press (OUP), 22 May 2023, [Peer-reviewed]
Scientific journal, Abstract

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common β chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

Zoobiquity experiments show the importance of the local MMP9-plasminogen axis in inflammatory bowel diseases in both dogs and patients
Takeshi Yamasaki, Noriyuki Nagata, Toru Atsumi, Rie Hasebe, Yuki Tanaka, Izuru Ohki, Shimpei Kubota, Yuta Shinohara, Yong Bin Teoh, Nozomu Yokoyama, Noboru Sasaki, Kensuke Nakamura, Hiroshi Ohta, Takehiko Katsurada, Yoshihiro Matsuno, Shintaro Hojyo, Shigeru Hashimoto, Mitsuyoshi Takiguchi, Masaaki Murakami
International Immunology, 35, 7, 313, 326, Oxford University Press (OUP), 18 Mar. 2023, [Peer-reviewed]
Scientific journal, Abstract

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn’s disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Dupuytren’s contracture-associated SNPs increase SFRP4 expression in non-immune cells including fibroblasts to enhance inflammation development
Hiroaki Kida, Jing-Jing Jiang, Yuichiro Matsui, Ikuko Takahashi, Rie Hasebe, Daisuke Kawamura, Takeshi Endo, Hiroki Shibayama, Makoto Kondo, Yasuhiko Nishio, Kinya Nishida, Yoshihiro Matsuno, Tsukasa Oikawa, Shimpei I Kubota, Shintaro Hojyo, Norimasa Iwasaki, Shigeru Hashimoto, Yuki Tanaka, Masaaki Murakami
International Immunology, 35, 7, 303, 312, Oxford University Press (OUP), 31 Jan. 2023, [Peer-reviewed]
Scientific journal, Abstract

Dupuytren’s contracture (DC) is an inflammatory fibrosis characterized by fibroproliferative disorders of the palmar aponeurosis, for which there is no effective treatment. Although several genome-wide association studies have identified risk alleles associated with DC, the functional linkage between these alleles and the pathogenesis remains elusive. We here focused on two single nucleotide polymorphisms (SNPs) associated with DC, rs16879765 and rs17171229, in secreted frizzled related protein 4 (SFRP4). We investigated the association of SRFP4 with the IL-6 amplifier, which amplifies the production of IL-6, growth factors and chemokines in non-immune cells and aggravates inflammatory diseases via NF-κB enhancement. Knockdown of SFRP4 suppressed activation of the IL-6 amplifier in vitro and in vivo, whereas the overexpression of SFRP4 induced the activation of NF-κB-mediated transcription activity. Mechanistically, SFRP4 induced NF-κB activation by directly binding to molecules of the ubiquitination SFC complex, such as IkBα and βTrCP, followed by IkBα degradation. Furthermore, SFRP4 expression was significantly increased in fibroblasts derived from DC patients bearing the risk alleles. Consistently, fibroblasts with the risk alleles enhanced activation of the IL-6 amplifier. These findings indicate that the IL-6 amplifier is involved in the pathogenesis of DC, particularly in patients harboring the SFRP4 risk alleles. Therefore, SFRP4 is a potential therapeutic target for various inflammatory diseases and disorders, including DC.

High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore
Kaoru Murakami, Shimpei I. Kubota, Kumiko Tanaka, Hiroki Tanaka, Keiichiroh Akabane, Rigel Suzuki, Yuta Shinohara, Hiroyasu Takei, Shigeru Hashimoto, Yuki Tanaka, Shintaro Hojyo, Osamu Sakamoto, Norihiko Naono, Takayui Takaai, Kazuki Sato, Yuichi Kojima, Toshiyuki Harada, Takeshi Hattori, Satoshi Fuke, Isao Yokota, Satoshi Konno, Takashi Washio, Takasuke Fukuhara, Takanori Teshima, Masateru Taniguchi, Masaaki Murakami
Lab on a Chip, 23, 22, 4909, 4918, Royal Society of Chemistry (RSC), 2023, [Peer-reviewed]
Scientific journal, Our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants including the next pandemic.

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, 22 Aug. 2022, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, 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, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, 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, Springer Science and Business Media LLC, Dec. 2021, [Peer-reviewed]
Scientific journal, 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, Aug. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, 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, (一社)日本癌学会, Oct. 2020
English

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, 120回, SF, 7, (一社)日本外科学会, Aug. 2020
Japanese

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, 24 Jun. 2020, [International Magazine]
English, Scientific journal, 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.

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, 日本癌学会, Sep. 2019
English

ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer
Shigeru Hashimoto, Shotaro Furukawa, Ari Hashimoto, Akio Tsutaho, Akira Fukao, Yurika Sakamura, Gyanu Parajuli, Yasuhito Onodera, Yutaro Otsuka, Haruka Handa, Tsukasa Oikawa, Soichiro Hata, Yoshihiro Nishikawa, Yusuke Mizukami, Yuzo Kodama, Masaaki Murakami, Toshinobu Fujiwara, Satoshi Hirano, Hisataka Sabe
Proceedings of the National Academy of Sciences of the United States of America, 116, 35, 17450, 17459, 27 Aug. 2019, [Peer-reviewed], [Lead author]
Scientific journal

Endothelin type B receptor interacts with the 78-kDa glucose-regulated protein.
Yuichi Mazaki, Tsunehito Higashi, Yasuhito Onodera, Jin-Min Nam, Ari Hashimoto, Shigeru Hashimoto, Takahiro Horinouchi, Soichi Miwa
FEBS letters, 593, 6, 644, 651, Mar. 2019, [Peer-reviewed], [International Magazine]
English, Scientific journal, Endothelin (ET)-1 is involved in the vascular system, cell proliferation and apoptosis. ET receptors consist of ET type A receptor (ETA R) and ET type B receptor (ETB R). ETA R and ETB R generally exhibit opposite responses, although many exceptions exist. In the present study, we attempted to identify ETA R- or ETB R-specific binding proteins to understand the differences in ETA R- and ETB R-mediated responses after ET-1 stimulation. The 78-kDa glucose-regulated protein (GRP78) showed a stronger binding affinity towards ETB R than towards ETA R. Moreover, GRP78 overexpression promoted ETB R-mediated ERK activation and GRP78 silencing suppressed ETB R-mediated ERK activation. Furthermore, ETB R can localize GRP78 to the cell periphery. These results suggest that the interaction of ETB R with GRP78 affects ERK activation and GRP78 localization.

GRP78 promotes ERK activation through endothelin type B receptor
Mazaki Yuichi, Higashi Tsunehito, Onodera Yasuhito, Nam Jin-Min, Hashimoto Ari, Hashimoto Shigeru, Horinouchi Takahiro, Miwa Soichi
Proceedings for Annual Meeting of The Japanese Pharmacological Society, 92, 1-P-110, Japanese Pharmacological Society, 2019
Japanese, 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.
Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Hirokazu Sugino, Tsukasa Oikawa, Hisataka Sabe
Cell communication and signaling : CCS, 16, 1, 94, 94, 04 Dec. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Arf6 and its ZEB1-EPB41L5 mesenchymal axis are required for both mesenchymal- and amoeboid-type invasion of cancer cells.
Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Hisataka Sabe
Small GTPases, 9, 5, 420, 426, 03 Sep. 2018, [International Magazine]
English, Scientific journal, 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.

Necessity of p53-binding to the CDH1 locus for its expression defines two epithelial cell types differing in their integrity.
Tsukasa Oikawa, Yutaro Otsuka, Yasuhito Onodera, Mei Horikawa, Haruka Handa, Shigeru Hashimoto, Yutaka Suzuki, Hisataka Sabe
Scientific reports, 8, 1, 1595, 1595, 25 Jan. 2018, [International Magazine]
English, Scientific journal, 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.

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 : CCS, 16, 1, 1, 1, 05 Jan. 2018, [International Magazine]
English, Scientific journal, 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.

Prognostic significance of erythrocyte protein band 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, 35, 9, 543.e17-543.e24, Sep. 2017, [International Magazine]
English, Scientific journal, 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-4N0M0) 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.

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, 5, 9, e259, Nature Publishing Group, Sep. 2016, [Peer-reviewed]
English, 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.
Hisataka Sabe, Ari Hashimoto, Shigeru Hashimoto, Tsukasa Oikawa
Molecular & cellular oncology, 3, 4, e1185564, Jul. 2016, [International Magazine]
English, Scientific journal, 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, Apr. 2016
Scientific journal

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, 10656, 08 Feb. 2016, [International Magazine]
English, Scientific journal, 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-Gα12 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.

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, 197, 7, 983, 996, Jun. 2012, [Peer-reviewed]
English, Scientific journal

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, Sep. 2011, [Peer-reviewed]
English, Scientific journal

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, Aug. 2011, [Peer-reviewed]
English, Scientific journal

乳癌細胞における恒常的なNF-kappaBの活性化に対するNIKおよびA20の役割(Roles for NIK and A20 in constitutive NF-kappaB activation in breast cancer cells)　　　　　　　　　　　　　　　
斉藤 愛記, 橋本 茂, 佐邊 壽孝, 山岡 昇司
日本癌学会総会記事, 68回, 323, 323, (一社)日本癌学会, Aug. 2009
English

EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition
Mariko Hirano, Shigeru Hashimoto, Shigenobu Yonemura, Hisataka Sabe, Shinichi Aizawa
JOURNAL OF CELL BIOLOGY, 182, 6, 1217, 1230, Sep. 2008, [Peer-reviewed]
English, Scientific journal

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, Apr. 2008, [Peer-reviewed]
English, Scientific journal

Fbx8 Makes Arf6 Refractory to Function via Ubiquitination
Hajime Yano, Itaru Kobayashi, Yasuhito Onodera, Fr{\'{e, e } }ric Luton, Michel Franco, Yuichi Mazaki, Shigeru Hashimoto, Kazuhiro Iwai, Ze{\textquotesingle}ev Ronai, Hisataka Sabe
Molecular Biology of the Cell, 19, 3, 822, 832, American Society for Cell Biology ({ASCB}), Mar. 2008, [Peer-reviewed]

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, 日本癌学会, Aug. 2007
English

CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery
Jin-Min Nam, Yasuhito Onodera, Yuichi Mazaki, Hiroyuki Miyoshi, Shigeru Hashimoto, Hisataka Sabe
EMBO JOURNAL, 26, 3, 647, 656, Feb. 2007, [Peer-reviewed]
English, Scientific journal

ArfGAP family proteins in cell adhesion, migration and tumor invasion
Hisataka Sabe, Yasuhito Onodera, Yuichi Mazaki, Shigeru Hashimoto
Current Opinion in Cell Biology, 18, 5, 558, 564, Elsevier {BV}, Oct. 2006, [Peer-reviewed]

Targeting AMAP1 and cortactin binding bearing an atypical src homology 3/proline interface for prevention of breast cancer invasion and metastasis
Shigeru Hashimoto, Mayumi Hirose, Ari Hashimoto, Masaki Morishige, Atsuko Yamada, Harumi Hosaka, Ken Ichi Akagi, Eiji Ogawa, Chitose Oneyama, Tsutomu Agatsuma, Masato Okada, Hidenori Kobayashi, Hiromi Wada, Hirofumi Nakano, Takahisa Ikegami, Atsushi Nakagawa, Hisataka Sabe
Proceedings of the National Academy of Sciences of the United States of America, 103, 18, 7036, 7041, 02 May 2006
Scientific journal

Neutrophil direction sensing and superoxide production linked by the GTPase-activating protein GIT2
Yuichi Mazaki, Shigeru Hashimoto, Tohru Tsujimura, Masaki Morishige, Ari Hashimoto, Kosuke Aritake, Atsuko Yamada, Jin-Min Nam, Hiroshi Kiyonari, Kazuki Nakao, Hisataka Sabe
Nature Immunology, 7, 7, 724, 731, Springer Nature, May 2006, [Peer-reviewed]

Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities.
Yasuhito Onodera, Shigeru Hashimoto, Ari Hashimoto, Masaki Morishige, Yuichi Mazaki, Atsuko Yamada, Eiji Ogawa, Masashi Adachi, Takaki Sakurai, Toshiaki Manabe, Hiromi Wada, Nariaki Matsuura, Hisataka Sabe
The EMBO journal, 24, 5, 963, 73, 09 Mar. 2005, [International Magazine]
English, Scientific journal, 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.
Shigeru Hashimoto, Ari Hashimoto, Atsuko Yamada, Yasuhito Onodera, Hisataka Sabe
Methods in enzymology, 404, 216, 31, 2005, [International Magazine]
English, Scientific journal, 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.
Kojima C, Hashimoto A, Yabuta I, Hirose M, Hashimoto S, Kanaho Y, Sumimoto H, Ikegami T, Sabe H
The EMBO journal, 23, 22, 4413, 4422, Oct. 2004, [Peer-reviewed]
English, Scientific journal

Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive.
Larisa Y Romanova, Shigeru Hashimoto, Kee-Oh Chay, Mikhail V Blagosklonny, Hisataka Sabe, J Frederic Mushinski
Journal of cell science, 117, Pt 17, 3759, 68, 01 Aug. 2004, [International Magazine]
English, Scientific journal, Tyrosine phosphorylation of paxillin regulates actin cytoskeleton-dependent changes in cell morphology and motility in adherent cells. In this report we investigated the involvement of paxillin tyrosine phosphorylation in the regulation of actin cytoskeleton-dependent polarization and motility of a non-adherent IL-3-dependent murine pre-B lymphocytic cell line Baf3. We also assessed the effect of phorbol myristate acetate (PMA), a phorbol ester analogous to those currently in clinical trials for the treatment of leukemia, on paxillin phosphorylation. Using tyrosine-to-phenylalanine phosphorylation mutants of paxillin and phosphospecific antibody we demonstrated that IL-3 stimulated phosphorylation of paxillin tyrosine residues 31 and 118, whereas the tyrosines 40 and 181 were constitutively phosphorylated. Phosphorylation of paxillin residues 31 and 118 was required for cell polarization and motility. In the presence of IL-3, PMA dramatically reduced the phosphorylation of residues 31 and 118, which was accompanied by inhibition of cell polarization and motility. This PMA effect was partially recapitulated by expression of exogenous tyrosine 31 and 118 mutants of paxillin. We also demonstrated that PMA inhibited the IL-3-induced and activation-dependent tyrosine phosphorylation of focal adhesion kinase. Thus, our results indicate that phosphorylation of paxillin tyrosine residues 31 and 118 regulates actin-dependent polarization and motility of pre-B Baf3 cells, both of which could be inhibited by PMA. They also suggest that inhibition of upstream signaling by PMA contributes to the decrease of paxillin phosphorylation and subsequent changes in cell morphology.

Requirement for Arf6 in breast cancer invasive activities.
Shigeru Hashimoto, Yasuhito Onodera, Ari Hashimoto, Miwa Tanaka, Michinari Hamaguchi, Atsuko Yamada, Hisataka Sabe
Proceedings of the National Academy of Sciences of the United States of America, 101, 17, 6647, 52, 27 Apr. 2004, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

A novel mode of action of an ArfGAP, AMAP2/PAG3/Papalpha, in Arf6 function
HASHIMOTO S
J Biol Chem, 279, 37677, 37684, 2004

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization
Yuichi Mazaki, Shigeru Hashimoto, Katsuya Okawa, Asako Tsubouchi, Kuniaki Nakamura, Ryohei Yagi, Hajime Yano, Akiko Kondo, Akihiro Iwamatsu, Akira Mizoguchi, Hisataka Sabe
Molecular Biology of the Cell, 12, 3, 645, American Society for Cell Biology ({ASCB}), Mar. 2001, [Peer-reviewed]

Interaction of Paxillin with p21-activated Kinase (PAK)
Shigeru Hashimoto, Asako Tsubouchi, Yuichi Mazaki, Hisataka Sabe
Journal of Biological Chemistry, 276, 8, 6037, American Society for Biochemistry {\&} Molecular Biology ({ASBMB}), Nov. 2000, [Peer-reviewed]

Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation
H Yano, H Uchida, T Iwasaki, M Mukai, H Akedo, K Nakamura, S Hashimoto, H Sabe
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 97, 16, 9076, 9081, Aug. 2000
English, Scientific journal

パキシリンのチロシンリン酸化を介する細胞-細胞間接触検知機構の解析　　　　　　　　　　　　　　　
矢野 元, 中村 邦明, 内田 浩, 橋本 茂, 佐邊 壽孝
日本癌学会総会記事, 58回, 234, 234, (一社)日本癌学会, Aug. 1999
Japanese

Mitosis specific serine phosphorylation and downregulation of one of the focal adhesion protein, paxillin
Ryuji Yamaguchi, Yuichi Mazaki, Kiichi Hirota, Shigeru Hashimoto, Hisataka Sabe
Oncogene, 15, 15, 1753, Springer Nature, Oct. 1997, [Peer-reviewed]

MACROPHAGE DIFFERENTIATION-SPECIFIC EXPRESSION OF NF-IL6, A TRANSCRIPTION FACTOR FOR INTERLEUKIN-6
S NATSUKA, S AKIRA, Y NISHIO, S HASHIMOTO, T SUGITA, H ISSHIKI, T KISHIMOTO
BLOOD, 79, 2, 460, 466, Jan. 1992, [Peer-reviewed]
English, Scientific journal

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

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

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, Sep. 2018
日本癌学会, English

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, Dec. 2017
生命科学系学会合同年次大会運営事務局, English

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], Dec. 2017
生命科学系学会合同年次大会運営事務局, Japanese

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, Sep. 2017
日本癌学会, English

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, Sep. 2017
日本癌学会, English

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, Oct. 2016
日本癌学会, English

メバロン酸経路阻害剤スタチンはArf6経路を高発現する癌に有効である　　　　　　　　　　　　　　　
橋本 あり, 橋本 茂, 及川 司, 大塚 勇太郎, 半田 悠, 小野寺 康仁, 佐邊 壽孝, 日本生化学会大会プログラム・講演要旨集, 89回, [1P, 268], Sep. 2016
(公社)日本生化学会, Japanese

膵癌細胞の浸潤・転移および化学療法抵抗性メカニズムの解明
古川聖太郎, 橋本あり, 橋本茂, 小野寺康仁, 及川司, 大塚勇太郎, 佐邊壽孝, 平野聡, 日本外科学会定期学術集会(Web), 116th, PS-002-2 (WEB ONLY), 002, Apr. 2016
(一社)日本外科学会, Japanese

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], Dec. 2015
(公社)日本生化学会, English

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, Sep. 2014
日本癌学会, English

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, Sep. 2014
日本癌学会, English

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, Sep. 2014
日本癌学会, English

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, 01 May 2014

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, 07 Oct. 2013

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, Oct. 2013
日本癌学会, English

細胞が持つリサイクルシステム研究の新展開 p53変異によるGEP100-Arf6-AMAP1経路の活性化と乳癌の浸潤形質獲得　　　　　　　　　　　　　　　
橋本 茂, 橋本 あり, 小根山 千歳, 吉河 歩, 杉野 弘和, 半田 悠, 芳野 正修, 大塚 勇太郎, 小野寺 康仁, 岡田 雅人, 佐邊 壽孝, 日本生化学会大会プログラム・講演要旨集, 86回, 2S04a, 3, Sep. 2013
(公社)日本生化学会, Japanese

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, Aug. 2012
日本癌学会, English

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, Aug. 2012
日本癌学会, English

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, Aug. 2012
日本癌学会, English

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, Aug. 2012
日本癌学会, English

変異p53が癌浸潤転移シグナル経路を創出し活性化する
橋本あり, 橋本茂, 吉河歩, 杉野弘和, 半田悠, 木下留美子, 畑中佳奈子, 三上修治, 谷野美智枝, 味藤静, 佐藤宏紀, 大塚勇太郎, 芳野日南子, 加戸由加里, NAM Jin‐Min, 小野寺康仁, 田中伸哉, 白土博樹, 佐邊壽孝, 日本分子生物学会年会プログラム・要旨集(Web), 35th, 2W10II-1 (WEB ONLY), 2012
Japanese

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, 2P-0166 (WEB ONLY), 2012
Japanese

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, Sep. 2011
日本癌学会, English

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, Sep. 2011
日本癌学会, English

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, Sep. 2011
日本癌学会, English

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, May 2011
(一社)日本細胞生物学会, English

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, May 2011
(一社)日本細胞生物学会, English

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, 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, Dec. 2010
(公社)日本生化学会, Japanese

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, Dec. 2010
(公社)日本生化学会, English

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, Dec. 2010
(公社)日本生化学会, English

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, Aug. 2010
日本癌学会, English

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, Aug. 2010
日本癌学会, English

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, Aug. 2010
日本癌学会, English

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, Aug. 2010
日本癌学会, English

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, Jun. 2010
English, Summary international conference

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, May 2010
(一社)日本細胞生物学会, English

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, Aug. 2009
English, Book review

蛋白質のリン酸化と細胞応答 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, 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, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 81回・31回, 4S2, 3, Nov. 2008
(公社)日本生化学会, Japanese

濃度勾配性因子による細胞極性・細胞運動の制御 組織構築、創傷治癒およびがんの浸潤・転移の機構解明を目指して 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, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 81回・31回, 4S14, 3, Nov. 2008
(公社)日本生化学会, Japanese

【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, 26, 15, 2349, 2355, Sep. 2008
(株)羊土社, Japanese

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, Sep. 2008
日本癌学会, English

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, Sep. 2008
日本癌学会, English

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, Jun. 2008
(一社)日本細胞生物学会, English

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, 2008

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, 4S14-3, 2008
Japanese

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, 佐邊壽孝, 生化学, 2P-0426, 2008
Japanese

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, Jan. 2008
English

Fbx8によるユビキチン化を介するArf6の抑制的制御と上皮組織形態形成との関連の可能性について(Fbx8 makes Arf6 refractory to function via ubiquitination: implication in epithelial tissue organization)　　　　　　　　　　　　　　　
矢野 元, 小野寺 康仁, 鳥井 郁子, 真崎 雄一, 橋本 茂, 辻村 亨, 佐邊 壽孝, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 80回・30回, 1T21, 3, Nov. 2007
(公社)日本生化学会, English

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, Aug. 2007
日本癌学会, English

Arf6をユビキチン化するE3リガーゼFbx8のがんにおける発現不全(Loss of Fbx8, a component of E3 ligase mediating Arf6 ubiquitination, in different human tumors)　　　　　　　　　　　　　　　
矢野 元, 真崎 雄一, 橋本 茂, 辻村 亨, 佐邊 壽孝, 日本癌学会総会記事, 66回, 142, 143, Aug. 2007
日本癌学会, English

Glioblastoma浸潤における低分子量G蛋白質Arf6の機能解析
森重真毅, 森重真毅, 橋本茂, 阿部竜也, 藤木稔, 古林秀則, 佐邊壽孝, 日本脳神経外科学会総会抄録集(CD-ROM), 66th, 2007

Expression of AMAP1 provides novel targets to inhibit the invasion of human glioma cells
Masaki Morishige, Shigeru Hashimoto, Tatsuya Abe, Hidenori Kobayashi, Hisataka Sabe, NEURO-ONCOLOGY, 8, 4, 435, 435, Oct. 2006
English, Summary international conference

乳癌細胞の浸潤におけるAMAP1のユビキチン化の役割(Property of AMAP1 to be monoubiquitinated via binding with CIN85 and Cbl is crucial for breast cancer invasive activity)　　　　　　　　　　　　　　　
Nam Jin-Min, 小野寺 康仁, 橋本 茂, 佐邊 壽孝, 日本癌学会総会記事, 65回, 295, 296, Sep. 2006
日本癌学会, English

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, 橋本 茂, 佐邊 壽孝, 日本癌学会総会記事, 65回, 311, 311, Sep. 2006
日本癌学会, Japanese

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, 古林 秀則, 橋本 あり, 魏 樹梅, 山田 敦子, 佐邊 壽孝, 日本癌学会総会記事, 65回, 456, 456, Sep. 2006
日本癌学会, Japanese

乳癌細胞におけるFbox8発現不全と浸潤性獲得の関連性
矢野元, 橋本茂, 佐邊壽孝, 佐邊壽孝, 日本癌学会学術総会記事, 65th, 2006

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, 2006

浸潤性乳癌細胞のAMAP1/コータクチン相互作用インターフェースの癌浸潤阻害剤の標的としての評価　　　　　　　　　　　　　　　
橋本 茂, 廣瀬 まゆみ, 橋本 あり, 森重 真毅, 山田 敦子, 小野寺 康仁, 小川 栄治, 和田 洋巳, 池上 貴久, 中川 敦史, 佐邊 壽孝, 日本癌学会総会記事, 64回, 309, 309, Sep. 2005
日本癌学会, Japanese

Arf6ユビキチン化機構と乳癌細胞浸潤性獲得過程との関連性　　　　　　　　　　　　　　　
矢野 元, 橋本 茂, 小野寺 康仁, 佐邊 壽孝, 日本癌学会総会記事, 64回, 173, 173, Sep. 2005
日本癌学会, Japanese

浸潤・転移・血管新生研究の進歩 乳癌における浸潤形質獲得過程　　　　　　　　　　　　　　　
佐邊 壽孝, 小野寺 康仁, ナム・ジンミン, 橋本 あり, 森重 真毅, 橋本 茂, 日本癌学会総会記事, 64回, 513, 513, Sep. 2005
日本癌学会, Japanese

Regulation of epithelial cell-cell interaction by Arf6 signaling
Koichi Miura, Hajime Yano, Yasuhito Onodera, Chie Kojima, Shigeru Hashimoto, Hisataka Sabe, CELL STRUCTURE AND FUNCTION, 30, 5, 5, Jun. 2005
English, Summary international conference

Novel regulation of Arf6 activity
Hajime Yano, Itaru Kobyashi, Shigeru Hashimoto, Hisataka Sabe, CELL STRUCTURE AND FUNCTION, 30, 49, 49, Jun. 2005
English, Summary international conference

乳癌細胞におけるArf6蛋白質の量的制御機構と浸潤性獲得　　　　　　　　　　　　　　　
矢野 元, 古林 格, 小野寺 康仁, 橋本 茂, 佐邊 壽孝, 日本癌学会総会記事, 63回, 265, 265, Sep. 2004
日本癌学会, Japanese

Arf6は乳癌細胞の浸潤性獲得において必須の分子装置の一つである　　　　　　　　　　　　　　　
橋本 茂, 小野寺 康仁, 橋本 あり, 森重 真毅, 田中 美和, 浜口 道成, 佐邊 壽孝, 日本癌学会総会記事, 63回, 265, 265, Sep. 2004
日本癌学会, Japanese

浸潤性乳癌細胞におけるAMAP1の発現と機能　　　　　　　　　　　　　　　
小野寺 康仁, 橋本 茂, 真崎 雄一, 橋本 あり, 森重 真毅, 松浦 成昭, 佐邊 壽孝, 日本癌学会総会記事, 63回, 267, 267, Sep. 2004
日本癌学会, Japanese

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, May 2004
English, Summary international conference

Tyrosine phosphorylation of paxillin α is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells
Kuniaki Nakamura, Hajime Yano, Hiroshi Uchida, Shigeru Hashimoto, Erik Schaefer, Hisataka Sabe, Hisataka Sabe, Hisataka Sabe, Hisataka Sabe, Journal of Biological Chemistry, 275, 27155, 27164, 01 Sep. 2000

細胞接着と細胞骨格の制御と細胞形態形成 細胞骨格制御におけるパキシリンと低分子量G蛋白質群との機能連関　　　　　　　　　　　　　　　
佐邊 壽孝, 橋本 茂, 近藤 明子, 坪内 朝子, 内田 浩, 中村 邦明, 矢野 元, 真崎 雄一, 生化学, 72, 8, 593, 593, Aug. 2000
(公社)日本生化学会, Japanese

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, 11, 4, 1315, 1327, Apr. 2000
English

ARF GAP活性を有するパキシリン結合性新規タンパク質　　　　　　　　　　　　　　　
近藤 明子, 橋本 茂, 真崎 雄一, 佐邊 壽孝, 日本癌学会総会記事, 58回, 184, 184, Aug. 1999
日本癌学会, Japanese

ARF6 GAP活性を示すパキシリン結合性タンパク質PAG3は細胞運動性を制御する
近藤明子, 橋本茂, 矢野元, 永山国昭, 真崎雄一, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 22nd, 1999

Paxillin function during epitherial-mesenchymal transdifferentiation
UCHIDA Hiroshi, YANO Hajime, MAZAKI Yuichi, HASHIMOTO Shigeru, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 01 Dec. 1998
Japanese

A novel protein that anchors a focal adhesion protein paxillin to the perinuclear regions
MAZAKI Yuichi, YANO Hajime, OKAWA Katsuya, HASHIMOTO Shigeru, IWAMATSU Akihiro, SABE Hisatake, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 01 Dec. 1998
Japanese

Analysis of the novel paxillin binding protein
KONDO Akiko, HASHIMOTO Shigeru, MAZAKI Yuichi, NAGAYAMA Kuniaki, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 01 Dec. 1998
Japanese

Epithelial cell survival signals
HASHIMOTO Shigeru, MAZAKI Yuichi, UCHIDA Hirosi, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 21, 0, 01 Dec. 1998
Japanese

膜裏打ち分子による細胞内情報伝達 上皮間充織形質転換と細胞生存性・運動性の制御 インテグリン裏打ち蛋白質パキシリン(Paxillin)を中心として　　　　　　　　　　　　　　　
佐邊 壽孝, 真崎 雄一, 内田 浩, 橋本 茂, 生化学, 70, 8, 703, 703, Aug. 1998
(公社)日本生化学会, Japanese

接着斑タンパク質パキシリン(Paxillin)のゴルジ装置への局在について　　　　　　　　　　　　　　　
真崎 雄一, 大川 克也, 内田 浩, 橋本 茂, 岩松 明彦, 佐邊 壽孝, 日本癌学会総会記事, 57回, 146, 146, Aug. 1998
日本癌学会, Japanese

ヒト癌におけるpaxillin isoformの発現の解析　　　　　　　　　　　　　　　
内田 浩, 真崎 雄一, 橋本 茂, 佐邊 壽孝, 日本癌学会総会記事, 57回, 451, 451, Aug. 1998
日本癌学会, Japanese

上皮系細胞のインテグリンを介する生存性維持機構に基付いた細胞癌化機構の解析　　　　　　　　　　　　　　　
橋本 茂, 真崎 雄一, 内田 浩, 佐邊 壽孝, 日本癌学会総会記事, 57回, 450, 450, Aug. 1998
日本癌学会, Japanese

Paxillim isoforms in mowse. Lack of the l islorm and developmeutally speciled βisoform expression.　　　　　　　　　　　　　　　
Jonrual of Biological chemistry, 273, 22435, 22441, 1998

Analysis of paxillin isoforms in mice.
真崎雄一, 橋本茂, 佐辺寿孝, 日本分子生物学会年会プログラム・講演要旨集, 20th, 1997

単球及び癌細胞でのパキシリンアイソフォームの発現と結合タンパク質の解析(共著)　　　　　　　　　　　　　　　
生化学雑誌, 272, 7432, 7444, 1997

Monocyte cells and cancer cells express novel paxillin isoforms with different binding
Journal of Biological Chemistry, 272, 11, 7437, 7444, 1997

Paxillin isoforms generated by exon-insertions exhibit altered expression and binding to focal adhesion proteins
MASAKI Yuichi, HASHIMOTO Shigeru, SABE Hisataka, 日本分子生物学会年会プログラム・講演要旨集, 19, 661, 661, 01 Aug. 1996
English

酵母TFIIDの変異体を用いた酸性転写活性化因子GAL4-VP16による転写活性化機構の解析(共著)　　　　　　　　　　　　　　　
自然, 369, 252, 255, 1994

Yeast TFIID point mutants defective in activation by acidic activator GAL4-VP16　　　　　　　　　　　　　　　
Nature, 369, 252, 255, 1994

インターロイキン6遺伝子転写調節因子(NF-IL6)(共著)　　　　　　　　　　　　　　　
化学免疫, 51, 299, 322, 1992

A NUCLEAR FACTOR FOR THE IL-6 GENE (NF-IL6)
S AKIRA, H ISSHIKI, T NAKAJIMA, S KINOSHITA, Y NISHIO, S HASHIMOTO, S NATSUKA, T KISHIMOTO, CHEMICAL IMMUNOLOGY, 51, 299, 322, 1992
English, Book review

RNA結合蛋白質Arid5aによる間葉形質及び免疫回避作動の連動性・多様性の研究
科学研究費助成事業
Apr. 2022 - Mar. 2025
橋本 茂
癌に関連した死亡の大部分は、転移によるものである。その本質は、癌のintrinsicな可塑性と共にextrinsicな腫瘍微小環境の多様性との連動性にある。癌の可塑性の分子機序として、上皮-間葉形質転換 (EMT)があげられる。EMTは上皮か間葉かのbinaryな変換ではなく両形質を含む可塑性に富んだ一過性の間葉形質維持状態として捉えられる。さらに、様々な癌種において、間葉形質と免疫回避との関連性が報告されている。
本研究は、癌の原発部位及び転移過程におけるRNA結合蛋白質Arid5aによる転写後mRNA安定化制御を介した間葉形質獲得と免疫抑制環境形成との連動性と多様性に着目し、それらの作用機序を解明することを目的とする。さらに、創傷治癒過程での可塑性誘導・終結における機能的役割との比較により、如何なる免疫制御の破綻が転移性癌の起源・多様化及び免疫抑制環境形成に繋がるのかに関しても検討を行う。
当該研究期間において、転移過程における間葉形質獲得と免疫抑制環境形成に関するArid5aの機能解析を進めた。EGFP及びLuciferaseを恒常的に発現させる為、当該遺伝子を同時に発現させるレンチウイルスベクターを構築し、野生型並びにArid5a遺伝子を欠損させたマウス膵癌細胞KPC及びマウス乳癌細胞4T1にinfectionし、目的の細胞株を樹立した。初めに、当該細胞群を尾静注転移モデルでの転移形成をIVIS in vivo imagingにより確認後、転移部位からsortingにより回収した野生型及びArid5a欠損細胞について、RNAseq.解析を進め、癌種間並びに両細胞間で発現の異なる遺伝子群を同定した。当該候補遺伝子に関してCLIP-RNAseq.解析によりArid5aが標的とする候補mRNAsを絞り込んだ。現在、それらの遺伝子群に関する機能解析を進めている。
日本学術振興会, 基盤研究(C), 北海道大学, 22K07203

Analysis of molecular bases of the relationship between mesenchymal phenotype and metabolic reprogram in clear cell renal cancer
Grants-in-Aid for Scientific Research
Apr. 2017 - Mar. 2020
Hashimoto Shigeru
We have previously shown that the Arf6 pathway is involved in the acquisition of invasiveness and drug-resistance of breast, renal, and pancreatic cancer. In this project, we demonstrated that the epigenetic regulator, EZH2, which has been reported to be statistically significantly associated with the poor prognosis of clear cell renal cell carcinoma (ccRCC), post-transcriptionally augments the protein expression levels of compounds of Arf6 pathway. Moreover, we found that Arf6 pathway is involved in the suppression of the amount of intracellular reactive oxygen species in ccRCC via activation of autophagy/mitophagy.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Osaka University, 17K07151

乳腺組織リモデリングにおける細胞運動性の統合的制御機構の解明
科学研究費助成事業
Apr. 2011 - Mar. 2013
橋本 茂
私共は、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根幹的役割を果たしていること、Arf6のエフェクターがAMAP1であること、EGFレセプター経路介した乳癌の浸潤形質獲得においてArf6を活性化因子がGEP100であることを明らかにした。さらに、GEP100-Arf6-AMAP1経路（以下Arf6経路）が、VEGFR2の活性化に伴う血管新生に関与することなど多様な細胞浸潤性誘導に関与することを見出した。当該研究期間において、AMAP1のエフェクター機能を解析する過程の中で、AMAP1がPRKD2を介してインテグリンと結合することを見出した。EGF刺激に伴ってArf6経路がRab5cと協調してインテグリンのリサイクルエンドソームから細胞膜への輸送に関与することを見出した。さらに、AMAP1がEMT誘導に伴って発現が誘導されるEPB41L5と特異的に結合することを見出し、Arf6経路の活性化に伴うinvadopodia形成あるいはE-cadherinを介した細胞-細胞間接着の消失が誘導される分子機序を見出した。また、AMAP1及びEPB41L5の発現が癌抑制遺伝子p53を介して抑制されることを明らかにした。p53欠損と、幹細胞様形質の誘導、あるいは、EMT誘導との関連が明らかとなっている。さらに、basal-likeに分類される乳癌の約80%において癌抑制遺伝子p53の遺伝子変異が見られ、その他の遺伝子変異がほとんど見られないことが報告されている。私共がこれまで高浸潤性乳癌細胞として用いていたすべての細胞が、basal-like乳癌と同じ遺伝子発現プロファイルを示すこと、さらに、p53に変異があることが明らかになっている。ことなどから、GEP100-Arf6- AMAP1経路が、乳腺組織幹細胞における細胞運動性・浸潤性の制御に関与している可能性が示唆された。
日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 23111501

Analysis of molecular mechanisms of the acquisition of tumor invasiveness
Grants-in-Aid for Scientific Research
2009 - 2012
HASHIMOTO Sigeru
We have previously shown that the GEP100-Arf6-AMAP1 pathway, activated by receptor tyrosine kinases, is involved in the acquisition of breast cancer invasiveness. Our analyses demonstrate that the activation of GEP100-Arf6-AMAP1 pathway via the c-Met/HGFR signaling is essential for the acquisition of the invasiveness of some breast cancer cells during hypoxia- or TGFss1-induced EMT. Moreover, we found that the expression of some molecules within the GEP100-Arf6-AMAP1 pathway are regulated by hypoxia-inducible factor (HIF) or EZH2, which have shown to be involved in the initiation and maintenance of the stemness in hypoxia.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 21370092

Analysis of molecular mechanisms of the acquisition of tumor invasiveness and metastasis
Grants-in-Aid for Scientific Research
2010 - 2011
HASHIMOTO Sigeru
In 1911, Otto Aichel postulated the concept of cell fusion as a mechanism of tumor malignancy. Recently, there are a lot of clinical and experimental discoveries of cell fusion as one of the driving forces of tumor progression. However, its molecular mechanisms remain to be elucidated. We have previously shown that GEP100-Arf6-AMAP1 signaling pathway contributes to the acquisition of invasiveness of some breast cancer cells. Here, we found that Arf6 and GEP100 were involved in the regulation of cell-cell fusion between highly invasive breast cancer cells, or bone marrow-derived cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, 22657045

Analysis of molecular mechanisms of Arf6 activation during acquisition of tumor invasiveness
Grants-in-Aid for Scientific Research
2007 - 2008
HASHIMOTO Shigeru
はじめ幾つかの癌において、レセプター型チロシンキナーゼであるEGFレセプターの高発現と、癌の悪性度との間に相関があることが知られている。しかし、癌の浸潤形質獲得に特異的なシグナル伝達経路があるか否かについては、不明のままであった。申請者らの研究成果から、シグナル分子GEP100がEGF レセプターを介したシグナル伝達経路と、乳癌の浸潤形質獲得に必須な低分子量G蛋白質Arf6の活性化とを結び付ける新規のシグナル伝達経路の存在が明らかとなった。さらに、病理学的解析から、このGEP100-Arf6経路が浸潤性乳癌の約80%に存在することが示唆された
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Osaka Bioscience Institute, 19570190

Analysis of molecular machinery for the acquisition of breast cancer invasiveness
Grants-in-Aid for Scientific Research
2005 - 2006
HASHIMOTO Shigeru
Our purpose is to elucidate the mechanism of acquisition of cancer cell invasiveness during tumor progression. Previously we found that a small GTPase, Arf6, and its effector, AMAP1, both play pivotal roles in the invasiveness of different breast cancer cells. We also showed that protein expression of Arf6 and AMAP1 in breast cancer cells correlate well with their invasive phenotypes, while expression in non-invasive cells and normal mammary epithelia is marginal.
During this research period, we obtained the following results:
1. Study of the molecular mechanisms regulating protein levels of Arf6 and AMAP1 in highly invasive breast cancer cells.
Arf6 and AMAP1 were ubiquitinated, but there was no correlation between their ubiquitination and their protein levels. On the other hand, in MDA-MB-231, AMAP1 protein levels were found to be translationally regulated through the mTOR pathway. We also found that AMAP1 is monoubiquitinated by Cbl and provide evidence that the ability of AMAP1 to be monoubiquitinated is important for its involvement in invasion.
2. Clarification of the signaling mechanisms of Arf6 activation in tumor invasion.
Expression or overexpression of several growth factor receptors, such as EGFR, ErbB2, and c-Met, often correlate with invasion and metastasis of different types of tumors. We identified that GEP100, but not other guanine nucleotide exchanging factors for Arf GTPases, is responsible for Arf6 activation to induce tumor invasion. GEP100 bound directly to phosphorylated EGFR to activate Arf6. Interfaces involved in the binding of GEP100 and EGFR may provide drug targets for tumor therapeutics.
3. Analysis of the correlation of the Arf6/AMAP1 pathway with invasiveness in other tumors.
We found that Arf6/AMAP1 signaling is also employed in the invasion of several kinds of cancer cell lines, derived from glioblastomas and lung tumors. We are now planning to perform immunohistochemical analyses of surgical specimens of these tumors.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Osaka Bioscience Institute, 17570166

Roles played by Arf6 in cell migration and invasion
Grants-in-Aid for Scientific Research
2004 - 2005
SABE Hisataka, HASHIMOTO Shigeru, MIURA Koichi, HASHIMOTO Ari
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 AMAP2 has a role in recruiting paxillin to sites of focal adhesions in epithelial cells. We have further demonstrated that AMAP1 is crucial for invasive activities of different breast cancer cells, in which AMAP1 functions by forming a trimeric protein complex with paxillin and cortactin. siRNA-mediated knockdown of AMAP1 effectively block invasive and metastatic activities of breast cancer cells.
Then, there was an enigma why GAP protein like AMAP1 can act as a necessary factor for tumor invasion, in spite of the simple idea that it may rather act to inhibit the invasion. Indeed, biochemical assays have shown a lack of efficient GAPing activity of AMAP1 against Arf6. We thus tested a mode of interaction between AMAP1 and Arf6, and found that AMAP1 via its ArfGAP domain binds to GTP-Arf6 stably, even in the presence of divalent cations. Binding of AMAP1 to other GTP bound Arfs, like Arf1 and Arf5, was negligible. We also conducted several cell biological assays, and concluded that AMAP1, and also AMAP2, act as effectors for GTP-Arf6. Both paxillin and cortactin are known to be essential for invadopodia formation, that are sites of tumor cell invasion into basement membranes. We showed that by binding to GTP-Arf6, AMAP1 has a role in recruiting paxillin and cortactin to invadopodia in breast cancer cells. Next we addressed to identify a GEF that is responsible for Arf6 activation in migration and invasion. We have succeeded in this identification, and also identified signaling pathways as to how this Arf6GEF becomes activated by extracellular stimuli. Besides these, we also showed that Arf6 can be ubiquitinated. We identified a E3 ligase involved in this ubiquitination, and shown that this is a non-canonical ubiquitination and that loss of the E3 ligase expression may contribute to invasive phenotypes tumor cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), OSAKA BIOSCIENCE INSTITUTE, 16370090

Cell motility and the backward bulk flow of the plasma membrane components
Grants-in-Aid for Scientific Research
2002 - 2003
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.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), OSAKA BIOSCIENCE INSTITUTE, 14380340

Role of paxillin-associatcd ARFGAPs in cell migration.
Grants-in-Aid for Scientific Research
2000 - 2001
SABE Hisataka, YAGI Ryohei, YANO Hajime, HASIMOTO Sigeru
ARF6 regulates endosomal recycling. We have shown that PAG3/Papα/KIAA0400 acts as a GTPase-activation protein (GAP) specific for ARF6.We study here molecular mechanims how PAG3 is involved in endosomal recycling to be an ARF6GAP. We found that PAG3, via its proline-rich region, binds to the src homology 3 (SH3) domain of several components of the endocytic machinery, and analysed its interaction with amphiphysin IIa. PAG3 existed at ARF6(Q67L)-positive membrane ruffles colocalized with amphyphysin Ha, but the majority exists at intracellular tubulovesicular structure. Overexpression of the amphiphysin ha SH3 domain is known to block endocytosis. Likewise, overexpression of the proline-rich region of PAG3 blocked both clathrin-dependent and independent endocytosis, while mutations of amino acids essential for the binding abolished such blockage. The SH3 domain of amphiphysin IIa. also binds to dynamin, a mechano-enzyme essential for the late step of endocytosis. We found that PAG3 exhibits almost one order of magnitude higher affinity than that of dynamin towards amphiphysin ha. We also demonstrated that PAG3 can be phosphorylated by a protein tyrosine kinase, Pyk2, but not by its close relative Fak ; and this phosphorylation inhibits the association with amphiphysin ha. With further results, we propose that PAG3 recruits amphiphysin ha to the plasma membrane, probably through interaction with the activity of GTP-bound Arf6 ; and external stimuli triggering endocytosis evoke tyrosine phosphorylation of PAG3, the phosphorylated PAG3 then releases amphiphysin ha to associate with components of endocytic machinery such as dynamin.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), OSAKA BIOSCIENCE INSTITUTE(OBI), 12480219

インテグリンを介する細胞接着シグナル制御と癌化細胞の生存性・侵潤能の解析
科学研究費助成事業
1999 - 2001
佐邊 壽孝, 真崎 雄一, 矢野 元, 橋本 茂, MARIUS Sudol, DONALD E. Ingber, JOAN S. Brugge
細胞が運動する際には、細胞の前方部にインテグリン接着点が新たに形成される。インテグリンは多くの種類の蛋白質をその細胞質領域に集積させることにより機能する。我々は、「このような裏打ち蛋白質群を集積させる過程が単に細胞質における自由拡散過程なのか、それとも、何か能動的な機序が存在するのか」との設問を立て、解析を開始した。
それまで機能解析をしてきたパキシリンというインテグリン裏打ち蛋白質に関して上記のような解析をしたところ、このものは、核周辺領域に細胞質プールが存在し、運動中に細胞前方に形成されるラミニポデアにパキシリンを集積させる過程は、細胞質での自由拡散ではなく、何らかの能動的な機序が存在することを強く示唆する結果を得た。そこで、パキシリンをプローブとしパキシリンの細胞内動態を説明できるような蛋白質を検索したところ、小胞/膜/蛋白質の輸送に関与する低分子性GTP結合蛋白質であるARF蛋白質に対するGAP(GTPase-activating protein)活性を持つ一連の興味深い一連の蛋白質が得られ、これらの蛋白質をPAGs(Paxillin-associated ARFGAP proteins)と命名した。現在、5種類のcDNAが得られており、その内、2種に関してほぼ初期段階の解析が終了し、論文に纏めている。ARFは哺乳類では6種のアイソフォームが存在するが、解析の終わった2種のPAG蛋白質はin vivoでそれぞれ異なったARFアイソフォームに対するGAP活性を示すこと、それらはパキシリンの細胞内での局在とダイナミックスの制御に互いに異なった機序により関与していること、さらには、細胞運動の制御にも関与していることを明らかにしている。特に、パキシリン結合性蛋白質として単離したこのようなPAG蛋白質が全てARFGAP活性を持っていることは、パキシリンのフォーカルコンタクトへの集積過程は、その過程自身に対して何らかの負のフィードバック制御機構を持っていることを示唆している。また、単球の成熟過程において発現誘導されるPAGが存在し、それは、ヒト動脈硬化病変における泡沫細胞に高レベル発現していることも観察している。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 11137310

インテグリンを介する細胞接着シグナル制御と癌化細胞の生存性・浸潤能の解析
科学研究費助成事業
2000 - 2000
橋本 茂, 中村 邦明, 矢野 元, 真崎 雄一, 八木 良平
細胞が運動する際、前方にfilopodiaとlamellipodiaが形成され、focal complexの形成を経て、細胞体の前方への移動に伴いインテグリンが集積したfocal adhesionが形成される。Rho-family低分子量G蛋白質の活性がこのような構造の形成や脱形成に深く関与していることは周知の通りである。また、最近、ARF-family低分子量G蛋白質がアクチン細胞骨格再構築に関与することも明らかにされている。ARF-family蛋白質は従来、主に細胞内での小胞輸送や膜輸送に関与することが示されていた分子である。
これまでに、インテグリン裏打ち蛋白質、パキシリンがゴルジ装置を含む核周辺部位にも局在していることを見い出していた。この局在がパキシリンのfilopodiaやlamellipodia構造への集積やインテグリン接着点形成過程に関連があると考え、生化学的/細胞生物学的解析を進めた。その結果、パキシリンが一連のARFGAP活性を持つ蛋白質と会合し、これらはパキシリンの核周辺局在とインテグリン接着点への集積、アクチン骨格構造制御、さらに、細胞運動性制御に関与することを明らかにした。
RacやCdc42の下流因子であるセリン/スレオニンリン酸化酵素PAK(p21-activated kinase)が、インテグリンシグナルや細胞骨格再構成に重要な役割を果たす事は良く知られている。調べた限りの幾つかの代表的な細胞接着斑蛋白質の中で、パキシリンのみが結合性を示した。PAKにはPIXやNck等、他に幾つかの結合蛋白質が知られているが、PAKとパキシリンとの結合はPAKとPIXやNckとの結合と競合的であった。さらに、パキシリンは不活性型及び、活性型PAKの両者に会合することができた。従って、パキシリンがPAKをfocal complexへアンカーする蛋白質であると考えられる。
日本学術振興会, 特定領域研究(C), (財)大阪バイオサイエンス研究所, 12218238

インテグリンを介した単球/マクロファージの細胞運動性制御機構の解析
科学研究費助成事業
1999 - 1999
佐邊 壽孝, 橋本 茂
成熟単球cDNA libraryをパキシリンをプロープとしfar-western法にてスクリーニングし、パキシリン結合性蛋白質cDNAを単離同定した。その結果、ARFGAPを示す新規蛋白質が得られた。我々は、成熟単球細胞以外でもパキシリン結合性蛋白質の精製やfar-western法による同定を行っているが、同様にARFGAP活性示す別の蛋白質が得られている。我々は、これら一群の蛋白質をPAGs(Paxillin-associated ARFGAP proteins)と命名し、今回成熟単球から得られたものをPAG3と命名した。
我々は、単球の成熟に伴ってパキシリンが発現誘導されチロシンリン酸化されることを明らかにしているが、PAG3もこの過程で発現誘導、チロシンリン酸化され、成熟単球において、主に細胞形質膜辺縁部にパキシリンを共局在した。ARFは哺乳類では6種のアイソフォームが存在し、小胞/膜/蛋白質の輸送やアクチン細胞骨格の再構成に関与することが知られている。PAG3はARF6に対するGAP(GTPase-activating protein)として機能することを明らかにすると共に、その強制発現は、パキシリンの細胞接着点への集積と細胞運動性とに対し、阻害的に働くことを明らかにした。さらに、PAG3はヒト動脈硬化病変における泡沫細胞に高レベル発現している可能性を示唆する観察結果も得ている。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 11158222

上皮細胞における細胞接着を介した生存性シグナルの解析
科学研究費助成事業
1999 - 1999
橋本 茂, 真崎 雄一, 佐邊 壽孝
上皮系細胞の生存性維持には増殖因子やサイトカインの刺激に加えて細胞接着シグナルが必要である。本研究では、インテグリン接着を介するシグナル伝達の機能的制御機構についてインテグリン裏打ちタンパク質パキシリンに着目した解析を進めている。今年度は、インテグリン接着点へのパキシリンの集積機構について解析を進めた。これまでに、パキシリンには核周辺領域に細胞質プールが存在し、運動中に細胞前方に能動的に集積させる機構が存在することを示唆させる結果を得ていた。この集積機構に関わる分子を解析するために、パキシリンに結合する分子群を同定し、想定される機能を持つ分子の検索を行った。その結果、ARF GAPモチーフを持つ一群のタンパク質を見い出した。その中の一つであるPAG3(Paxillin-associated ARF GAP protein 3)は、マクロファージ様に分化したU937細胞のcDNAからパキシリンをプローブとしたファーウエスタン法により見い出した。PAG3は、未分化の単球では細胞質に存在しているが、単球が接着性を獲得すると発現が亢進し、細胞辺縁部へ局在し、パキシリンと共局在することが観察された。また、PAG3のARF GAP活性がパキシリンの接着点への局在に重要な役割を果たしていること、分化した単球細胞の運動性の制御に関わっていることを示唆させる結果を得た。従って、パキシリンの細胞内局在は、単なる自由拡散ではなくARFの活性が関与する制御機構によって規定されていることが示された。また、ヒトの粥状動脈硬化病変においてマクロファージの特徴を示す泡沫化細胞にPAG3が強く発現していることを観察した。PAG3が病変の進展と関連した機能を有する可能性が示唆される。さらに、パキシリン及びその結合タンパク質がアクチン細胞骨格を制御する分子群と相互作用することを見い出した。細胞接着シグナルによる細胞骨格再構築の時間的/空間的制御機構に重点を置いた解析を開始している。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 11139271

Role of paxillin isoforms and their tyrosine phosphorylation in cell migration.
Grants-in-Aid for Scientific Research
1998 - 1999
SABE Hisataka, MAZAKI Yuichi, YANO Hajime, HASHIMOTO Sigeru
Temporal and spatial regulation of actin-based cytoskeletal organization and focal adhesion formation play an essential role in cell migration. We found that tyrosine phosphorylation of paxillin and pi3OCas was a prominent event upon integrin activation during epithelial-mesenchymal transdifferentiation and cell migration. Tyrosine phosphorylation of p130^ has been demonstrated to facilitate cell migration. We showed that tyrosine phosphorylation of paxillin cc acts to reduce haptotactic cell migration as well as transcellular invasive activities in several different experimental cell systems, whereas tyrosine phosphorylation of p130^ exerts opposing effects to those of paxillin
a. Each of the phosphorylation-null mutant acted as dominant-negatives for each phenotype. Moreover, we found that overexpression of paxillin a reduced the cell saturation density of NMuMG cells while overexpression of pi30^ increased it. These effects also seemed to be dependent on the tyrosine phosphorylation events. Cell growth rates and morphologies at growing phases were not significantly altered, nor were cells transformed. Addition of epidermal growth factor increased saturation density of the paxillin α-overexpressing cells, while no further increment was observed in p130^-overexpressing cells. We propose that tyrosine phosphorylation of paxillin a and p130^ exert opposing effects on several integrin-mediated cellular events, possibly through different signaling pathways. We also found that paxillin binds to several ARFGAPs. ARFGAPs are regulators of intracellular membrane trafficking. We currently analysing role of paxillin from aspects of its tyrosine phosphorylation and its interaction with ARFGAPs, in regulation of cell migratory activity.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., OSAKA BIOSCIENCE INSTITUTE(OBI), 10480199

単球・マクロファージ細胞の接着及び貧食過程におけるパキシリンの機能解析
科学研究費助成事業
1998 - 1998
左邊 壽孝, 矢野 元, 橋本 茂
上皮細胞や内皮細胞、さらには分化した単球/マクロファージ細胞等の接着性細胞にとって、細胞接着はその生存性の維持に必須である。接着が維持されないと、これらの細胞はanoikisと呼ばれる、速やかなprogrammed ce11 deathに陥る。インテグリンの活性化以降、phosphatidyl3kinase(PI3K)からAkt/protein kinaseBを経て、最終的にはcaspaseの活性抑制に至る経路が現在提唱されるに至っている。
しかし、肝心のインテグリンの直下因子でありP13Kの活性化を引き起こす蛋白質因子は同定されていない。以前には、これはFocal adhesion kinaseであると提唱されていたが、我々が幾つかの検討を行ったところ、Focal adhesion kinaseではないことが判明した。さらに検討を進めたところ、Focal adhesion kinaseと似た分子量(120-140kDa)のチロシンリン酸化蛋白質がインテグリンの活性化に伴ってPI3KのSH2(N)領域に結合することが明らかになった。同様な分子量のPI3K結合性蛋白質は、調べた限りanoikisを起こす全ての細胞に認められた。既知の、分子量が120-140kDaのチロシンリン酸化蛋白質に対する抗体で、入手可能なものすべてを検討したが、それらのいずれでもなかった。そこで、現在この蛋白質の精製、単離同定を行っている。この因子が単球細胞の分化や、炎症部位での作業終了後の生存性の制御にどのように関わっているのか興味深い。また、上皮組織の癌化過程においてどのように関与するのかも注目される。
日本学術振興会, 特定領域研究(A), (財)大阪バイオサイエンス研究所, 10177213

癌細胞の浸潤形質獲得過程の解明　　　　　　　　　　　　　　　
Competitive research funding

Analysis of the regulation mechanisms for the acquisition of invasive phenotype of tumor cells　　　　　　　　　　　　　　　
Competitive research funding