Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation
Takayuki Sakurai, Norio Takei, Yangxuan Wei, Marina Hayashi, Akiko Kamiyoshi, Hisaka Kawate, Satoshi Watanabe, Masahiro Sato, Takayuki Shindo
Scientific Reports, 14, 1, Springer Science and Business Media LLC, 05 Dec. 2024
Scientific journal

Genome-wide CRISPR screen identifies MAD2L1BP and ANAPC15 as targets for brentuximab vedotin sensitivity in CD30+ peripheral T-cell lymphoma.
Keito Suto, Norio Takei, Keito Yokoyama, Masahiro Chiba, Takashi Ishio, Michiyuki Maeda, Hideki Goto, Tomoyuki Endo, Takanori Teshima, Yibin Yang, Masao Nakagawa
Leukemia, 21 Oct. 2024, [International Magazine]
English, Scientific journal

Abundant extrasynaptic expression of α3β4-containing nicotinic acetylcholine receptors in the medial habenula–interpeduncular nucleus pathway in mice
Asuka Tsuzuki, Miwako Yamasaki, Kohtarou Konno, Taisuke Miyazaki, Norio Takei, Susumu Tomita, Michisuke Yuzaki, Masahiko Watanabe
Scientific Reports, 14, 1, Springer Science and Business Media LLC, 20 Jun. 2024
Scientific journal, Abstract

Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)–interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and β4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3β4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and β4 subunits in the MHb–IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and β4 subunits disappeared in α5β4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb–IPN pathway suggests that α3β4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3β4-containing nAChRs in the crucial pathway of nicotine dependence.

Corrigendum to ‘CCL8 deficiency in the host abrogates early mortality of acute graft-versus-host disease in mice with dysregulated IL-6 expression’ [Experimental Hematology 2022; 106: 47-57]
Keita Igarashi, Norio Takei, Tsukasa Hori, Masaki Yamamoto, Hitoshi Sohma, Nobuhiro Suzuki, Hiroyuki Tsutsumi, Yukihiko Kawasaki, Yasuo Kokai
Experimental Hematology, 127, 70, 70, Elsevier BV, Nov. 2023
Scientific journal

Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.
Masahiro Chiba, Joji Shimono, Takashi Ishio, Norio Takei, Kohei Kasahara, Reiki Ogasawara, Takahide Ara, Hideki Goto, Koh Izumiyama, Satoko Otsuguro, Liyanage P Perera, Hiroo Hasegawa, Michiyuki Maeda, Satoshi Hashino, Katsumi Maenaka, Takanori Teshima, Thomas A Waldmann, Yibin Yang, Masao Nakagawa
Blood, 140, 18, 1951, 1963, 03 Aug. 2022, [International Magazine]
English, Scientific journal, Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark for ATLL pathogenesis. However, the mechanisms by which ATLL cells evade NK-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using two ATLL derived cell lines and discovered CD48 as one of the best enriched genes whose knockout conferred resistance to YT-1 NK cell line mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK cell effector function was confirmed using human primary NK cells with reduced IFNg induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCL) also expressed lower levels of CD48 than normal T-cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK cell mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK cell-associated immunotherapies.

Novel Interleukin-6 Inducible Gene PDZ-Binding Kinase Promotes Tumor Growth of Multiple Myeloma Cells.
Akinobu Ota, Ichiro Hanamura, Sivasundaram Karnan, Shingo Inaguma, Norio Takei, Vu Quang Lam, Shohei Mizuno, Jo Kanasugi, Md Wahiduzzaman, Md Lutfur Rahman, Toshinori Hyodo, Hiroyuki Konishi, Shinobu Tsuzuki, Hiroshi Ikeda, Akiyoshi Takami, Yoshitaka Hosokawa
Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research, 40, 8, 389, 405, Aug. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, [Figure: see text] Multiple myeloma (MM) remains an intractable hematological malignancy, despite recent advances in anti-MM drugs. Here, we show that role of PDZ binding kinase (PBK) in MM tumor growth. We identified that interleukin-6 (IL-6) readily increases PBK expression. Kaplan-Meier analysis showed that the MM patients with higher expression of PBK have a significant shorter survival time compared with those with moderate/lower expression of PBK. Knockout of PBK dramatically suppressed in vivo tumor growth in MM cells, while genome editing of PBK changing from asparagine to serine substitution (rs3779620) slightly suppresses the tumor formation. Mechanistically, loss of PBK increased the number of apoptotic cells with concomitant decrease in the phosphorylation level of Stat3 as well as caspase activities. A novel PBK inhibitor OTS514 significantly decreased KMS-11-derived tumor growth. These findings highlight the novel oncogenic role of PBK in tumor growth of myeloma, and it might be a novel therapeutic target for the treatment of patients with MM.

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.
Sakurai T, Kamiyoshi A, Takei N, Watanabe S, Sato M, Shindo T
Scientific reports, 9, 1, 9923, Jul. 2019, [Peer-reviewed]

ERO1α is a novel endogenous marker of hypoxia in human cancer cell lines.
Takei N, Yoneda A, Kosaka M, Sakai-Sawada K, Tamura Y
BMC cancer, 19, 1, 510, 510, May 2019, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Endoplasmic reticulum oxidase 1 alpha is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells
Mizuki Fujii, Akihiro Yoneda, Norio Takei, Kaori Sakai-Sawada, Marina Kosaka, Kenjiro Minomi, Atsuro Yokoyama, Yasuaki Tamura
JOURNAL OF BIOLOGICAL CHEMISTRY, 292, 38, 15649, 15660, Sep. 2017, [Peer-reviewed]
English, Scientific journal

Hypoxia-inducible ERO1a alpha promotes cancer progression through modulation of integrin-beta 1 modification and signalling in HCT116 colorectal cancer cells
Norio Takei, Akihiro Yoneda, Kaori Sakai-Sawada, Marina Kosaka, Kenjiro Minomi, Yasuaki Tamura
SCIENTIFIC REPORTS, 7, 1, 9389, Aug. 2017, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Spatiotemporal Regulation of Hsp90-Ligand Complex Leads to immune Activation
Yasuaki Tamura, Akihiro Yoneda, Norio Takei, Kaori Sawada
FRONTIERS IN IMMUNOLOGY, 7, 201, May 2016, [Peer-reviewed]
English

Generation and analysis of Olfactory receptor Knock-out and Knock-in mice
武井則雄, 太田明伸, 桜井敬之, 日本ゲノム編集学会大会要旨集, 8th, 2023

癌におけるERO1αの新規内在性低酸素マーカーとしての有効性の検証
武井則雄, 米田明弘, 澤田香織, 小坂まりな, 田村保明, 臨床ストレス応答学会大会抄録集, 13th, 2018

がん細胞進化を支える小胞体常在ストレス応答性分子の機能解析とこれを標的とする治療戦略
田村保明, 米田明弘, 武井則雄, 澤田香織, 臨床ストレス応答学会大会抄録集, 13th, 2018

HSP47によるトリプルネガティブ乳癌の転移能獲得機序の解明
米田明弘, 武井則雄, 澤田香織, 小坂まりな, 小坂まりな, 田村保明, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018

トリプルネガティブ乳癌の転移能におけるHSP47の作用機序の解明
米田明弘, 武井則雄, 澤田香織, 小坂まりな, 田村保明, 臨床ストレス応答学会大会抄録集, 13th, 2018

癌細胞におけるHSP47のIRE1α活性調節機構
米田明弘, 武井則雄, 澤田香織, 小坂まりな, 小坂まりな, 味呑憲二郎, 田村保明, 臨床ストレス応答学会大会抄録集, 12th, 2017

ケモカインCCL8の移植後早期発現は同種造血細胞移植における非再発死亡率の増加と相関する
山本雅樹, 山本雅樹, 堀司, 堀司, 畠山直樹, 五十嵐敬太, 五十嵐敬太, 稲澤奈津子, 鈴木信寛, 武井則雄, 伊藤陽一, 松本公一, 加藤剛二, 堤裕幸, 小海康夫, 日本造血細胞移植学会総会プログラム・抄録集, 39th, 2017

ERO1αは低酸素環境下においてintegrin-β1の立体構造形成に関与し癌細胞増殖に寄与する
武井則雄, 米田明弘, 澤田かおり, 小坂まりな, 小坂まりな, 味呑憲二郎, 田村保明, 臨床ストレス応答学会大会抄録集, 12th, 2017

ER01αはコラーゲンの分泌とMT1-MMPの生合成を介して肝星細胞の増殖を調節する
米田明弘, 藤井瑞希, 藤井瑞希, 武井則雄, 澤田香織, 横山敦郎, 田村保明, 臨床ストレス応答学会大会抄録集, 11th, 2016

肝星細胞における小胞体酸化還元酵素ERO1αの機能解析
米田明弘, 藤井瑞希, 藤井瑞希, 武井則雄, 澤田香織, 横山敦郎, 田村保明, 日本分子生物学会年会プログラム・要旨集(Web), 39th, 2016

ヒト肝星細胞における小胞体常在分子シャペロンの機能解析
藤井瑞季, 藤井瑞季, 藤井瑞季, 米田明弘, 武井則雄, 澤田香織, 横山敦郎, 飯田順一郎, 田村保明, 臨床ストレス応答学会大会抄録集, 10th, 2015

ニホンウナギのアロマターゼおよび3β-水酸基脱水素酵素の生殖腺における発現解析
風藤行紀, 小原真由子, 武井則雄, 井尻成保, 足立伸次, 山内晧平, 日本水産学会大会講演要旨集, 2008, 2008

癌・精巣特異的嗅覚受容体を介した癌転移機構の解明と臨床応用
科学研究費助成事業
01 Apr. 2023 - 31 Mar. 2027
武井 則雄, 太田 明伸, 桜井 敬之
日本学術振興会, 基盤研究(C), 北海道大学, 23K07801

Examination of the therapeutic effect on inflammatory bowel disease using HSPB6-positive exosomes derived from amnion-derived MSCs
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2021 - 31 Mar. 2024
桂田 武彦, 山本 幸司, 大西 俊介, 武井 則雄
本研究では、羊膜MSCから産生されるHSPB6陽性エクソソームを用いた抗炎症効果の詳細を細胞実験ならびに炎症性腸疾患誘導性動物モデルを用いて、未だ適切な治療法がないヒト炎症性腸疾患に対する臨床応用への基盤を整える。本研究では、以下の通り、HSPB6過剰発現ならびにHSPB6欠損羊膜MSCの作成およびそれらエクソソームの樹立することを明らかにした。羊膜MSCのHSPB6過剰発現株ならびに遺伝子欠損株を樹立する。HSPB6過剰発現株の作成には、CMVプロモーター下にHSPB6遺伝子を挿入したプラスミドをMSCに導入後、薬剤選択による選別を行い、得られたクローンはウエスタンブロット法による野生型およびmockコントロールとの比較解析から、有意に発現増加の認められるクローンを選別することで樹立した。HSPB6欠損株の作成にはCRISPR/Cas9システムを採用し、ヒトHSPB6エクソン領域に特異的なgRNAを設計し、CAGプロモーター下でCas9およびT3プロモーター下でgRNAを発現するプラスミドを構築、MSCに導入し、genotypingによる標的遺伝子座での塩基の欠損が認められ、ウエスタンブロット法で標的分子の翻訳消失が認められるクローンを選別した。結果、20個のクローン中4～5個程度の目的クローンが選別することができた。さらに、その細胞からエクソソームを精製し、HSPB6の発現について確認したところ、HSPB6過剰発現株ではMOCKに比べてHSPB6の発現が増加していた一方、HSPB6欠損株では、HSPB6の発現が消失していることを確認した。これらのことから、本研究において、HSPB6過剰発現株ならびにHSPB6欠損発現株が樹立され、さらに両株から産生される目的となるエクソソームも精製することができた。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 21K07952

Establishment of the platform for a drug discovery of multiple myeloma by targeting PBK-related signaling pathway
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2021 - 31 Mar. 2024
太田 明伸, 武井 則雄, 花村 一朗
多発性骨髄腫（Multiple myeloma， 以下MM）は形質細胞由来の難治性血液悪性腫瘍である。MMの臨床症状は多彩であり、治療やその効果の予測は難しい。臨床症状の原因として、細胞増殖の制御機構に異常をきたしたMM細胞の異常増殖が考えられているが、その分子学的機序には不明な点が多い。代表者は、MMの新規予後不良因子としてPDZ-binding kinase（以下PBK）を発見し、PBKがMM細胞の造腫瘍性に密接に関連することを示した。そこで本研究では、PBKを標的とした分子標的薬ならびにPBK関連分子との相互作用阻害がMMの新しい治療薬として有効である可能性を検証する方針とした。
はじめに、PBK遺伝子の生理的作用を見出す目的で、PBK遺伝子破壊（PBK-KO）マウスを樹立した。PBK-KOマウスは、正常に出産・発育し外見上特別な表現型を示さなかった。そこで、PBKが豊富に存在する精巣からRNAを抽出して、cDNAマイクロアレイ法とGene Set Enrichment Analysisにより遺伝子発現に関わる解析に着手した。
代表者は、公共のデータベースと遺伝子発現解析を利用したサブ解析から、PBKの発現レベルと高い相関性を示す遺伝子X（仮称）を見出した。遺伝子Xはインターロイキン６によって遺伝子発現が誘導され、その高発現はMM患者の生存率を有意に低下させること、MM細胞株の増殖に関連することを明らかにした。また、ゲノム編集法による内在性タグの導入と、タグ抗体を用いた免疫沈降によるプロテオミクス解析を実施し、遺伝子Xと相互作用する分子の同定を試みている。
さらに、PBKを高発現するMM細胞に対して増殖抑制効果の高い薬剤や化合物を同定するために、1500個のカスタム化合物ライブラリーを入手した。この化合物とPBKの発現を欠失させたアイソジェニッククローンを用いて、各種化合物に対する感受性の変化を解析中である。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Aichi Medical University, 21K08426

Analyses of functional differentiation and interaction of RAMP1,2 and 3 applying the maternal Cas9-based multiple genome editing
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2019 - 31 Mar. 2022
SAKURAI TAKAYUKI
The purpose of this study was to analyze the functional differentiation of RAMPs by analyzing DSS-induced colitis models, colorectal organoids, and macrophage cells using mice with mutations in RAMP subisoforms produced by our systematic multiple simultaneous genome editing methods. We found that RAMP1 and 3 are mainly involved in colitis in mice, and colitis increases in RAMP1-deficient mice, while RAMP3-deficient mice increased their resistance. Furthermore, we showed that RAMP1/RAMP3 signals respond to inflammatory signals simultaneously and synergistically in the inflammatory-stimulated colorectal epithelium, while RAMP1/RAMP3 signals respond to inflammatory signals simultaneously and inversely in macrophage cells.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Shinshu University, 19K07477

Elucidation of the mechanism of action of the olfactory receptor involved in cancer-specific metabolism
Grants-in-Aid for Scientific Research
01 Apr. 2018 - 31 Mar. 2021
Takei Norio
In glucose metabolism, cancer cells are known to have a predominant energy metabolism by glycolysis, even in an aerobic environment, as compared to normal cells. This property has been suggested to be associated with malignant tumors of cancer in various stress environments within the tumor.
In this study, we focused on the characteristic metabolic pathways of cancer that differ from these normal cells and conducted a study about the role of a orphan receptor, which is thought to be involved in its metabolism, and the verification of therapeutic effect of its inhibition. This result suggested that this molecule may be involved in the regulation of major metabolic enzymes in cancer-specific energy metabolic pathways, and expected to be a novel target molecule in cancer treatment.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 18K07060

Investigation of molecular mechanism by which PBK contributes to tumorigenesis in myeloma cells
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2018 - 31 Mar. 2021
Ota Akinobu
Multiple myeloma (MM) is a complex plasma cell neoplasm, accounting for approximately 10% of all hematological malignancies. Novel molecular-targeted therapy based on their genomic abnormalities in the patients with MM is really wanted to improve their clinical outcome. In this study, we identified that interleukin-6 readily increases PBK expression. Kaplan-Meier analysis showed that the MM patients with higher expression of PBK have a significant shorter survival time compared with those with moderate/lower expression of PBK. Knockout of PBK dramatically suppressed in vivo tumor growth in MM cells. Mechanistically, loss of PBK increased the number of apoptotic cells with concomitant decrease in the phosphorylation level of Stat3. A novel PBK inhibitor OTS514 significantly decreased KMS-11-derived tumor growth. These findings highlight the novel oncogenic role of PBK in tumor growth of myeloma, and it might be a novel therapeutic target for the treatment of patients with MM.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Aichi Medical University, 18K08342

Uncovering the molecular mechanisms of OR7C1 in cancer biology.
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
01 Apr. 2016 - 31 Mar. 2018
Takei Norio
OR7C1 is a G protein coupled receptor and expressed in cancer stem cell and suggested the possibility as a novel factor of poor prognosis. However, the function in cancer remain unclear. In this study, to uncover the importance of OR7C1 expression in cancer biology, we generated conventional OR7C1 knockout clones by using the CRISPR/Cas9 system. As a result, OR7C1 KO decreased tumorigenicity and metastasis in vivo and cell proliferation under specific medium conditions in vitro. In conclusion, our findings suggest that OR7C1 is predominantly activated under specific condition such as starvation and contributes to cancer progression. Collectively, OR7C1 could be used as both a diagnostic biomarker of cancer exacerbation and a therapeutic target.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, 16K20874

CCL8 KOマウスにおけるGVHD消失の分子機構解明に向けた基礎的研究
科学研究費助成事業 若手研究(B)
01 Apr. 2014 - 31 Mar. 2016
武井 則雄
前年度の研究結果を踏まえ、KOマウスのGVHD発症機構と関連の予想される主要組織適合抗原複合体(MHC)の発現等を含めたphenotype解析の追加実験および、in vitroでのallo recognitionにおけるサイトカイン産生などをWTとKOマウスで比較検証することでKOマウスを用いたGVHDマウスモデル解析におけるGVHD病態変化の要因を探索した。前年度からの本研究成果をまとめ現在論文投稿準備中である。
日本学術振興会, 若手研究(B), 札幌医科大学, 26870464