研究者詳細 - 江口　傑徳

論文 - 江口　傑徳

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Multiple Targeting of HSP Isoforms to Challenge Isoform Specificity and Compensatory Expression. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 141 - 161 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heat shock proteins (HSPs) are molecular chaperones that assist in protein folding, trafficking, and metabolism. Intracellular chaperone functions of HSPs had been well-investigated, but extracellular and exosomal HSPs have been recently found. Exosomal HSPs are intercellularly transferred, while extracellular HSPs play cytokine-like roles called chaperokines. We have shown that exosomal HSPs play key roles in intercellular communication between tongue carcinoma and tumor-associated macrophages in the tumor microenvironment. Notably, HSP90 isoforms consist of HSP90alpha, HSP90beta, mitochondrial TRAP1, and GRP94 in the endoplasmic reticulum. Moreover, many pseudogenes of HSP90 can be transcribed into RNA. Besides, the function of HSP90 is defined by their cochaperones, such as CDC37 or AHA1. Therefore, isoform-specific small interfering RNA (siRNA) is necessary for precisely targeting each HSP90 isoform and cochaperone. Nevertheless, we often encountered compensatory expression of HSP90 isoforms in the knockdown studies. Here, we provide dual and triple knockdown methods to target multiple RNA for challenging isoform-specific roles and compensatory expression of intracellular, extracellular, and exosomal HSPs.

DOI： 10.1007/978-1-0716-3342-7_12

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Proteomic Profiling of the Extracellular Vesicle Chaperone in Cancer. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 233 - 249 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Molecular chaperones are widely distributed intracellular proteins that play essential roles in maintaining proteome function by assisting in the folding of client proteins. Molecular chaperones, such as heat shock proteins (HSPs), are found intracellularly and extracellularly. Extracellular vesicles (EVs), such as exosomes, contain HSPs and horizontally transfer the functional chaperones into various recipient cells. Besides, mass spectrometry has enabled a comprehensive analysis of exosomal and EV proteins, which is useful in basic biomedical research to clinical biomarker search. We have performed deep proteome analysis of EVs, including exosomes, from metastatic tongue and prostate cancers and detected >700 protein types, including cytoplasmic, ER, mitochondrial, small, and large HSPs. Here, we provide protocols for isolating exosomes/EVs and deep proteome analysis to detect the EV chaperone.

DOI： 10.1007/978-1-0716-3342-7_18

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Large-Scale Databases and Portals on Cancer Genome to Analyze Chaperone Genes Correlated to Patient Prognosis. 招待査読国際誌

Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 293 - 306 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Molecular chaperones, such as heat shock proteins (HSPs), have attracted attention as molecules involved in malignant events in cancers and are potential therapeutic targets and biomarkers for tumor therapy. Furthermore, mutations in chaperones can significantly impact cancer risk and prognosis. Bioinformatics is a particularly useful method for developing biomarkers as a practical consideration for the immediate clinical application of data. Many large-scale databases and portals on cancer genome are nowadays publicly available, including the International Cancer Genome Consortium (ICGC); The Cancer Genome Atlas (TCGA), renamed as Genomic Data Commons (GDC); Catalogue of Somatic Mutations in Cancer (COSMIC); and Cancer Cell Line Encyclopedia (CCLE). Referring to these databases, advanced web portals are publicized, including cBioPortal, Human Protein Atlas (HPA), Kaplan-Meier (KM) plotter, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), Genomics of Drug Sensitivity in Cancer (GDSC), and Dependency Map (DepMap). Here, we assemble these databases and portals to clarify what is available and useful for current cancer research and provide protocols to utilize the HPA, KM plotter, and GEPIA2 for studies on chaperone genes in cancer patients. Utilizing these portals will reveal the correlation between tumor subtype-specific high expression of chaperone genes and patient prognosis. Our protocols are useful to increase systematic awareness of chaperones and find new biomarkers for diagnosis and prognosis and new targets for anticancer drugs.

DOI： 10.1007/978-1-0716-3342-7_22

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Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter. 招待査読国際誌

Andrew Ackerman, Toshiki Kijima, Takanori Eguchi, Thomas L Prince

Methods in molecular biology (Clifton, N.J.) 2693 1 - 11 2023年

記述言語：英語掲載種別：研究論文（学術雑誌）

The heat shock response (HSR) is a cellular mechanism for counteracting acute proteotoxic stress. In eukaryotes, transcriptional activation of the HSR is regulated by heat shock factor 1 (HSF1). Activation of HSF1 induces the expression of heat shock proteins (HSPs) that function as molecular chaperones to fold and maintain the three-dimensional structure of misfolded proteins. The regulation of the degree and duration of the HSR is controlled by multiple biochemical mechanisms that include posttranslational modification of HSF1 and numerous protein-protein interactions. In this chapter, we describe a method to evaluate the activation and deactivation of the HSR at the transcriptional level using a short half-life luciferase reporter assay. This assay can be used to further characterize the HSR or as a screen for small molecule inducers, amplifiers, or repressors.

DOI： 10.1007/978-1-0716-3342-7_1

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Protocol for CRISPR/Cas Genome Editing for Investigating Cell Communication Network. 招待査読国際誌

Yuka Okusha, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2582 157 - 167 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

The Cellular Communication Network Factor (CCN) family is composed of six members: CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3. The second member, CCN2/CTGF is a matricellular protein that promotes extracellular matrix (ECM) synthesis and controls angiogenesis. On the other hand, moonlighting/matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme that also functions as an intracellular transcription factor. Importantly, extracellular MMP3 is uptaken into cells, translocating into nuclei, and transcriptionally activating CCN2/CTGF gene in cancer and chondrocytes. Thus, the MMP3-CTGF axis balances the matrix metabolism and turnover in the tissue and tumor microenvironments. We established an MMP3 knockout cell line using the CRISPR/Cas9 system, demonstrating the sequential regulatory events of the MMP3-CCN2 axis in the microenvironment. Notably, our protocol is useful for generation of CCN knockout cells as well. Here we serve a protocol of the CRISPR/Cas9-based gene targeting in cultured cells for investigating cellular communication network.

DOI： 10.1007/978-1-0716-2744-0_11

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Transfection, Spinfection, Exofection, and Luciferase Assays for Analysis of CCN Genes Expression Mechanism. 招待査読国際誌

Takanori Eguchi, Yanyin Lu, Eman A Taha, Yuka Okusha

Methods in molecular biology (Clifton, N.J.) 2582 103 - 126 2023年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cell communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is protein inducible in response to TGFβ/Smad signal or the transcriptional activity of matrix metalloproteinase 3 (MMP3). We discovered that MMP3 in exosomes is transferable to recipient cells and then translocates into cell nuclei to transactivate the CCN2/CTGF gene. Exosomes and liposomes enable molecular transfection to recipient cells in vitro and in vivo. These small vesicles are surrounded by lipid membranes and carry proteins, RNA, DNA, and small chemicals. Here we define the exosome-based transfection as "exofection." In addition, spinfection increases the efficiencies of transfection, exofection, and viral infection, thus being compatible with various molecular transfer protocols. Here, we provide protocols, tips, and practical examples of transfection, spinfection, exofection, fluorescence microscopy, and luciferase assays to analyze the CCNs gene expression mechanisms.

DOI： 10.1007/978-1-0716-2744-0_9

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Comprehensive Method for Exosome Isolation and Proteome Analysis for Detection of CCN Factors in/on Exosomes. 招待査読国際誌

Takanori Eguchi, Yuka Okusha, Yanyin Lu, Kisho Ono, Eman A Taha, Shiro Fukuoka

Methods in molecular biology (Clifton, N.J.) 2582 59 - 76 2023年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.

DOI： 10.1007/978-1-0716-2744-0_6

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Western Blot Protocols for Analysis of CCN Proteins and Fragments in Exosomes, Vesicle-Free Fractions, and Cells. 査読国際共著国際誌

Kisho Ono, Yuka Okusha, Manh Tien Tran, Koki Umemori, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2582 39 - 57 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.

DOI： 10.1007/978-1-0716-2744-0_5

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Cdc37 as a Co-chaperone to Hsp90 招待査読国際誌

Thomas L Prince, Benjamin J Lang, Yuka Okusha, Takanori Eguchi, Stuart K Calderwood

Sub-cellular biochemistry 101 141 - 158 2023年

Multiplex Immunostaining Method to Distinguish HSP Isoforms in Cancer Tissue Specimens. 招待査読国際誌

Hotaka Kawai, Kisho Ono, Takanori Eguchi

Methods in molecular biology (Clifton, N.J.) 2693 281 - 291 2023年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heat shock proteins (HSPs) are often expressed in all nucleated cells, but their expression profiles differ. In particular, HSP90α and HSP90β have high sequence identity and have not been fully examined for their individual and compensatory functions as molecular chaperones, differences in client proteins, and extracellular distributions with exosomes. Immunohistochemical staining is a technique to visualize the presence and localization of target antigens using specific antibodies, of which the multiplex immunostaining method can reveal differences in protein expression in the same tumor tissue and the localization of proteins of interest within tumor tissue or single cells. The common multiplex immunostaining method uses multiple secondary antibodies of different reacting animal species to identify and detect different antigens, thus requiring different animals to be immunized with each primary antibody. Furthermore, the fluorescent-antibody method is the predominant multiplex staining method but has the critical disadvantage that permanent specimens cannot be prepared. Here, we outline a multiplex staining method for HSP90α and HSP90β based on the enzyme-antibody method that allows permanent specimens to be prepared without the restriction of immunized animal species.

DOI： 10.1007/978-1-0716-3342-7_21

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Roles of Cancer Exosomes in Immunosuppression and Immune Evasion

Mona Sheta, Eman Ahmed Taha, Yanyin Lu, Takanori Eguchi

Preprints 2022110004 2022年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Extracellular vesicles (EV), including exosomes and microvesicles, are released from various cells and alter recipient cell phenotypes and fates by their biomolecules. Here we review current knowledge about tumor EVs and how they prompt malignant cell communication with tumor-associated cells, such as cancer-associated fibroblasts, tumor endothelial cells, and immune cells. We delineate the major pathways and molecular players that influence each step of cancer initiation, progression, and resistance. Of note, cancer exosomes involve immunosuppression by tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells. Moreover, tumor exosomes can induce the apoptosis of killer T cells and immune checkpoint of dendritic cells and attenuate natural killer cells. An in-depth understanding of EV biology is essential to ensure the clinical development of exosome/EV-based therapeutic products, which will be of benefit to exosome manipulation in cancer management.

DOI： 10.20944/preprints202211.0004.v1

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SCAND1 Reverts EMT and Suppresses Tumor Growth and Collective Migration

Takanori Eguchi, Eva Csizmadia, Thomas L. Prince, Barbara Wegiel, Stuart K. Calderwood

Preprints 2022年10月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Epithelial-mesenchymal transition (EMT) is a reversible cellular program that transiently places epithelial (E) cells into pseudo-mesenchymal (M) cell states. The malignant progression and resistance of many types of carcinomas depends on EMT activation, partial EMT and hybrid E/M status in neoplastic cells. EMT is activated by tumor microenvironmental TGF&beta; signal and EMT-inducing transcription factors, such as ZEB1/2 in tumor cells. However, reverse EMT factors are less studied. We demonstrate that transcription factor SCAND1 can revert mesenchymal and hybrid E/M phenotype of cancer cells to a more epithelial, less invasive status and inhibit their proliferation and migration. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and transcriptional co-repression of target genes. We found that SCAND1-MZF1 co-expression and interaction correlated with maintaining epithelial features, whereas the simultaneous loss of SCAND1 and MZF1 correlated with mesenchymal features of tumor cells. Overexpression of SCAND1 over endogenous MZF1 in DU-145 prostate cancer cells reverted their hybrid E/M status into cobblestone morphology with increased epithelial adhesion by E-cadherin and &beta;-catenin relocation. Consistently, co-expression analysis in TCGA PanCancer Atlas revealed that both SCAND1 and MZF1 co-express and are negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBR, in prostate tumor specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, SCAND1-overexpressing DU-145 cells migrated slower than control cells with decreased lymph node metastasis of prostate cancer in a mouse tumor xenograft model. Kaplan-Meyer analysis showed high expression of MZF1 and SCAND1 to correlate with better prognoses in pancreatic cancer and head and neck cancers, although with poorer prognosis in kidney cancer. Overall, these data suggest that the combination of SCAND1-MZF1 complexes may revert the EMT mechanism in cancer to establish an epithelial phenotype. These effects seem to include co-repression of EMT-driver genes and suppression of tumor cell proliferation via inhibition of the MAP3K-MEK-ERK signaling pathway.

DOI： 10.20944/preprints202210.0475.v1

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HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts. 査読国際誌

Manh Tien Tran, Yuka Okusha, Kaung Htike, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Cell biochemistry and function 40 ( 8 ) 838 - 855 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90α and HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed that HSP90, a novel effector protein of Rab11b, modulated Rab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

DOI： 10.1002/cbf.3745

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口腔癌間質細胞由来CCL2による骨髄由来細胞動員への関与(Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment)

河合穂高, 冨田秀太, 江口傑徳, 大原利章, 小野喜章, 長塚仁

日本癌学会総会記事 81回 P - 3186 2022年9月

口腔癌間質由来のCCL2はCCR2陽性骨髄由来免疫抑制細胞の腫瘍間質への動員に関与する

河合穂高, メイ・ワトウ, 高畠清文, 冨田秀太, 小野喜章, 江口傑徳, 大原利章, 中野敬介, 長塚仁

日本がん免疫学会総会プログラム・抄録集 26回 91 - 91 2022年6月

Rab34 plays a critical role as a bi-directional regulator of osteoclastogenesis 査読国際誌

Yunxia Feng, Manh Tien Tran, co-first author, Yanyin Lu, Kaung Htike, Yuka Okusha, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Cell Biochemistry and Function 40 ( 3 ) 263 - 277 2022年3月

Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment. 査読国際誌

May Wathone Oo, Hotaka Kawai, Kiyofumi Takabatake, Shuta Tomida, Takanori Eguchi, Kisho Ono, Qiusheng Shan, Toshiaki Ohara, Saori Yoshida, Haruka Omori, Shintaro Sukegawa, Keisuke Nakano, Kuniaki Okamoto, Akira Sasaki, Hitoshi Nagatsuka

JCI insight 7 ( 1 ) 3186 2022年1月

Porphyromonas gingivalisはマクロファージの細胞外小胞を介して胎盤・胎児の成長発育を阻害する(Porphyromonas gingivalis impairs placental and fetal development through macrophage-derived extracellular vesicles)

棚井あいり, 福原瑶子, 江口傑徳, 河合穂高, 池亀美華, 岡村裕彦, 植田幸嗣

Journal of Oral Biosciences Supplement 2021 205 - 205 2021年10月

A novel role of HSP90 in regulating osteoclastogenesis by abrogating Rab11b-driven transport. 査読国際誌

Manh Tien Tran, Yuka Okusha, Yunxia Feng, Chiharu Sogawa, Takanori Eguchi, Tomoko Kadowaki, Eiko Sakai, Takayuki Tsukuba, Kuniaki Okamoto

Biochimica et biophysica acta. Molecular cell research 1868 ( 10 ) 119096 - 119096 2021年9月

Extracellular Hsp90 and protection of neuronal cells through Nrf2 査読国際誌

Stuart K Calderwood, Thiago J Borges, Takanori Eguchi, Benjamin J Lang, Ayesha Murshid, Yuka Okusha, Thomas L Prince

Biochemical Society Transactions 49 ( 5 ) 2299 - 2306 2021年8月

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