Kawahara, Ryota

写真a

Affiliation

Faculty of Science and Technology, Department of Applied Chemistry (Yagami)

Position

Assistant Professor (Non-tenured)/Research Associate (Non-tenured)/Instructor (Non-tenured)

Related Websites

External Links
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Profile Summary 【 Display / hide 】

  • We conduct basic research to elucidate the pathogenesis of diseases and to develop therapeutic drugs. In particular, we focus on "vasculogenic mimicry" and "glycosylation," which are biological phenomena closely related to malignant transformation of cancer, and analyze their mechanisms using chemical approaches.

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Career 【 Display / hide 】

  • 2020.04
    -
    2022.03

    JSPS Research Fellowship for Young Scientists DC2

  • 2024.04
    -
    Present

    Keio University, Department of Applied Chemistry, Faculty of Science and Technology, Research associate

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Academic Background 【 Display / hide 】

  • 2013.04
    -
    2017.03

    Keio University, Faculty of Science and Technology, Department of Applied Chemistry

    University, Graduated

  • 2017.04
    -
    2019.03

    Keio University, 理工学研究科

    Graduate School, Completed, Master's course

  • 2019.04
    -
    2022.03

    Keio University, 理工学研究科

    Graduate School, Completed, Doctoral course

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Academic Degrees 【 Display / hide 】

  • Ph.D., Keio University, Coursework, 2022.03

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Research Areas 【 Display / hide 】

  • Life Science / Molecular biology

  • Life Science / Cell biology

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Research Keywords 【 Display / hide 】

  • がん生物学

  • ケミカルバイオロジー

  • 糖鎖生物学

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Papers 【 Display / hide 】

  • Total Synthesis of Isodaphlongamine H by Iridium-Catalyzed Reductive [3 + 2] Cycloaddition of N-Hydroxylactam

    Iwamoto S., Nakano R., Sasaki K., Kobayashi S., Taira Y., Takei K., Kawakita R., Tokuyama A., Nakamura H., Tomoike M., Kawahara R., Murase A., Simizu S., Chida N., Okamura T., Sato T.

    Angewandte Chemie International Edition  2025

    ISSN  14337851

    　View Summary

    The total synthesis of isodaphlongamine H based on a lactam strategy, which enables quick access to complex cyclic amines, is described. The strategy begins with alkylation of a chiral lactam and subsequent N-oxidation via an imino ether to afford the N-hydroxylactam. For the key transformation to functionalize the amide carbonyl, an iridium-catalyzed reductive [3 + 2] cycloaddition of the N-hydroxylactam provides a tricyclic isoxazolidine in a one-pot process. After the coupling reaction with an allylic silane fragment, the total synthesis is accomplished through intramolecular Hosomi–Sakurai allylation to construct a pentacyclic core. The deoxygenated pentacyclic intermediate shows higher cytotoxicity against HeLa and U937 cell lines than isodaphlongamine H, and might become a lead compound for further biological study.

  • Identification of C-mannosylation in a receptor tyrosine kinase AXL

    Mori K., Suzuki T., Waki U., Hayashi S., Kadono S., Kawahara R., Takeuchi M., Mizuta H., Dohmae N., Katayama R., Simizu S.

    Glycobiology 34 ( 11 )  2024.11

    ISSN  09596658

    　View Summary

    C-mannosylation is a unique type of glycosylation in which a mannose is added to tryptophan in a protein. However, the biological function of C-mannosylation is still largely unknown. AXL is a receptor tyrosine kinase, and its overexpression contributes to tumor malignancy. The role of AXL in cancer cells is broad, including invasion, drug resistance, and vasculogenic mimicry formation. Although Trp320 of AXL was predicted to be C-mannosylated, it has not been confirmed. Here, we demonstrated that Trp320 of AXL is C-mannosylated, measured by mass spectrometry of recombinant AXL purified from various cancer cells. Furthermore, re-expression of C-mannosylation-deficient AXL in human breast cancer MDA-MB-231 cells lacking AXL by the CRISPR/Cas9 system resulted in reduction of vasculogenic mimicry formation. Interestingly, phosphorylation levels of AKT in C-mannosylation-deficient AXL re-expressing cells were comparable to those of parental and wild-type AXL re-expressing cells. These results represent the first discovery of C-mannosylation in a receptor tyrosine kinase and the possibility that C-mannosylation may affect AXL function, distinct from its downstream signaling in cancer cells.

  • DPY19L3 promotes vasculogenic mimicry by its C-mannosyltransferase activity

    Hassan Baydoun, Yuji Kato, Hiroki Kamo, Anna Hüsch, Hayato Mizuta, Ryota Kawahara, Siro Simizu

    Oncology Research (Oncology Research)  32 ( 4 ) 607 - 614 2024

    Joint Work, Accepted,  ISSN  09650407

    　View Summary

    C-mannosylation is a post-translational modification that occurs intracellularly in the endoplasmic reticulum. In humans, biosynthesis of C-mannosylation in proteins containing thrombospondin type 1 repeat is catalyzed by the DPY19 family; nonetheless, biological functions of protein C-mannosylation are not yet fully understood, especially in tumor progression. Vasculogenic mimicry (VM) is the formation of fluid-conducting channels by highly invasive and genetically deregulated tumor cells, enabling the tumors to form matrix-embedded vasculogenic structures, containing plasma and blood cells to meet the metabolic demands of rapidly growing tumors. In this study, we focused on DPY19L3, a C-mannosyltransferase, and aimed to unravel its role in VM. Knockout of DPY19L3 inhibited the formation of VM in HT1080 human fibrosarcoma cells. Re-expression of wild-type DPY19L3 recovered VM formation; however, DPY19L3 isoform2, an enzymatic activity-defect mutant, did not restore it, suggesting that the C-mannosyltransferase activity of DPY19L3 is crucial to its function. Furthermore, the knockdown of DPY19L3 in MDA-MB-231 breast cancer cells hindered its network formation ability. Altogether, our findings suggest that DPY19L3 is required for VM formation and stipulate the relevance of C-mannosylation in oncogenesis.

  • Biogenesis of fibrils requires C-mannosylation of PMEL

    Ryota Kawahara, Tomoko Usami, Satoko Arakawa, Hiroki Kamo, Takehiro Suzuki, Ryosuke Komatsu, Hiroyuki Hara, Yuki Niwa, Erina Shimizu, Naoshi Dohmae, Shigeomi Shimizu, Siro Simizu

    FEBS Journal (FEBS Journal)  290 ( 22 ) 5373 - 5394 2023.11

    Joint Work, Lead author, Accepted,  ISSN  1742464X

    　View Summary

    Premelanosome protein (PMEL), a melanocyte-specific glycoprotein, has an essential role in melanosome maturation, assembling amyloid fibrils for melanin deposition. PMEL undergoes several post-translational modifications, including N- and O-glycosylations, which are associated with proper melanosome development. C-mannosylation is a rare type of protein glycosylation at a tryptophan residue that might regulate the secretion and localization of proteins. PMEL has one putative C-mannosylation site in its core amyloid fragment (CAF); however, there is no report focusing on C-mannosylation of PMEL. To investigate this, we expressed recombinant PMEL in SK-MEL-28 human melanoma cells and purified the protein. Mass spectrometry analyses demonstrated that human PMEL is C-mannosylated at multiple tryptophan residues in its CAF and N-terminal fragment (NTF). In addition to the W153 or W156 residue (CAF), which lies in the consensus sequence for C-mannosylation, the W104 residue (NTF) was C-mannosylated without the consensus sequence. To determine the effects of the modifications, we deleted the PMEL gene by using CRISPR/Cas9 technology and re-expressed wild-type or C-mannosylation-defective mutants of PMEL, in which the C-mannosylated tryptophan was replaced with a phenylalanine residue (WF mutation), in SK-MEL-28 cells. Importantly, fibril-containing melanosomes were significantly decreased in W104F mutant PMEL-re-expressing cells compared with wild-type PMEL, observed using transmission electron microscopy. Furthermore, western blot and immunofluorescence analysis suggested that the W104F mutation may cause mild endoplasmic reticulumretention, possibly associated with early misfolding, and lysosomal misaggregation, thus reducing functional fibril formation. Our results demonstrate that C-mannosylation of PMEL is required for proper melanosome development by regulating PMEL-derived fibril formation.

  • Cofilin promotes vasculogenic mimicry by regulating the actin cytoskeleton in human breast cancer cells

    Minami Nakajima, Ryota Kawahara, Siro Simizu

    FEBS Letters (FEBS Letters)  597 ( 8 ) 1114 - 1124 2023.04

    Joint Work, Accepted,  ISSN  00145793

    　View Summary

    Vasculogenic mimicry (VM) is the formation of microvascular channels by cancer cells. VM requires cellular processes that are regulated by changes in cellular migration and morphology. Cofilin (CFL), a key regulator of actin depolymerization, has been reported to affect malignant phenotypes of cancer. We show that treatment with inhibitors of actin dynamics suppresses VM in MDA-MB-231 human breast cancer cells. We established CFL-knockout (KO) MDA-MB-231 cells and found that VM was attenuated in CFL-KO cells. Although the re-expression of wild-type CFL restored VM in CFL-KO cells, inactive phosphomimetic CFL failed to do so. Collectively, our results demonstrate that CFL is a critical regulator of VM and implicate CFL as a novel therapeutic target for breast cancer.

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Papers, etc., Registered in KOARA 【 Display / hide 】

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

  • がん細胞の血管擬態におけるマーカータンパク質の同定

    2020.04
    -
    2022.03

    Grants-in-Aid for Scientific Research, Grant-in-Aid for JSPS Fellows, No Setting

    　View Summary

    本研究の概要は、がん悪性化に寄与する血管擬態について、その存在や進行度の指標となるマーカータンパク質を同定することである。血管擬態とは、がん細胞自身が血管のように擬態することで腫瘍内血管を作り出し、腫瘍成長のエネルギー源となる血液の循環を促す現象である。そのため、血管擬態形成時に特異的に発現するタンパク質を同定し、それをバイオマーカーとして応用できれば、がんの予後診断や治療法選択を改善させられることが期待できる。本研究では、培養細胞を用いた実験により血管擬態のマーカータンパク質を同定することを目指す。
    がん細胞の血管擬態におけるマーカータンパク質を探索するため、3つの遺伝子に焦点を当て、それらが血管擬態形成を制御し得るか否かを調べた。
    ErbB4は、がん遺伝子として有名なEGFRやHER2と同じファミリーに属する遺伝子である。しかし、ErbB4とがんの関係については明確でないのが現状である。そこで、ヒト乳がん細胞株を用いて解析を行った結果、ErbB4が血管擬態形成を抑制することが明らかになった。一方で、リガンド刺激や遺伝子変異によりErbB4を活性化させると、血管擬態形成を促進させることが示された。さらに、EGFR阻害剤として知られているアファチニブを細胞に処理することで、ErbB4を介した血管擬態形成が効果的に抑制できることを示した。これらの結果により、ErbB4は血管擬態を正負に調節することでがんの進行に関与することが明らかになった。
    MetAP2は血管新生に寄与する遺伝子として知られているが、血管擬態における役割は分かっていない。そこで、阻害剤処理や遺伝子ノックアウトにより解析した結果、MetAP2の阻害によりヒトがん細胞の血管擬態が著しく抑制されることが明らかになった。
    Tyrosinaseはメラニン産生において必要不可欠な酵素であるが、メラノーマにおける役割はほとんど分かっていない。そこで、メラノーマ細胞においてtyrosinase遺伝子を欠損させた結果、血管擬態形成が促進した。また、色素欠乏症で報告されたアミノ酸変異が、tyrosinaseの血管擬態制御能に影響を与えることを示した。
    以上のように、がん細胞の種類によって血管擬態を制御する遺伝子が異なり、各々の遺伝子は血管擬態のマーカーとして有望である可能性が示された。また、これらの遺伝子は既に阻害剤も開発されているため、がんの治療標的としても有望である。
    令和3年度が最終年度であるため、記入しない。
    令和3年度が最終年度であるため、記入しない。

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Awards 【 Display / hide 】

  • 第80回日本癌学会学術総会　JCA若手研究者ポスター賞

    2021.10, 日本癌学会, ErbB4 はヒト乳がん細胞の血管擬態を負に調節する

    Type of Award： Award from Japanese society, conference, symposium, etc.

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Courses Taught 【 Display / hide 】

  • LABORATORY IN SCIENCE

    2025

  • LABORATORIES IN APPLIED CHEMISTRY D

    2025

  • LABORATORY IN SCIENCE

    2024

  • LABORATORIES IN APPLIED CHEMISTRY D

    2024

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Memberships in Academic Societies 【 Display / hide 】

  • 日本糖質学会, 

    2024
    -
    Present

  • 日本がん分子標的治療学会, 

    2020
    -
    Present

  • 日本癌学会, 

    2017
    -
    Present

  • 日本がん転移学会, 

    2017
    -
    Present
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