Springer Science and Business Media LLC Acta Medica Okayama 1478-811X 24 1 2026 MMP-3 cleavage of Lamin A induces pro-migratory nuclear deformity, nucleophagy, and their autophagic secretion with extracellular vesicles in metastatic cancer 146 EN Takanori Eguchi Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman A. Taha Department of Biochemistry, Faculty of Science, Ain Shams University Keisuke Nakano Department of Oral Pathology and Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Vikas Tiwari Council of Scientific & Industrial Research-Indian Institute of Toxicological Research Katsuki Takebe Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomohiro Inoue Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Lizi Xing Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Food and Health Sciences, Faculty of Environmental Studies, Hiroshima Institute of Technology Kuniaki Okamoto Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. MMPs have also been found in extracellular vesicles (EVs), such as exosomes. MMP-3 promotes tumor growth, epithelial-to-mesenchymal transition, genome instability, migration, invasion, and metastasis of cancer cells, and nuclear MMP-3 controls gene transcription. Intranuclear proteolysis by MMPs may significantly alter cancer progression. However, the nuclear substrates of MMP-3 have not been well investigated. In this study, we performed proteomic analyses to identify the nuclear substrates and EV proteins regulated by MMP-3. While rabidly metastatic colon cancer (LuM1) three-dimensionally cultured tumoroids secreted EVs containing 30 protein types, including Lamin A (LMNA), MMP-3, fibronectin (FN1), HSPA8 (Hsc70), ��-actin (ACTB), and vimentin (VIM), CRISPR/Cas9-based knockout of MMP-3 reduced the secretion of these proteins in EVs. Notably, EV-bound cleaved Lamin secretion was confirmed by immunoelectron microscopy. Also, MMP-3 formed proteolytic dimers via its hemopexin-like repeat domains in nuclei. Many nuclear MMP-3-binding proteins, including Lamin A/C, histones, topoisomerases, and hnRNPs, were screened by co-immunoprecipitation followed by proteomics. Proteolytic MMP-3 overexpression generated a C-terminal 30-kDa fragment of Lamin A, whose cleavage site was defined via structural analysis. MMP-3 digestion of Lamin A induced nuclear deformity (atypia) required for cell migration in confined space. The cleaved Lamin A and MMP-3 were transported with autophagosomes (LC3B+), nucleophagosomes, and amphisomes (CD63 + LC3B+) and co-secreted with EVs. Proteolytic MMP-3 also induced nuclear speckles of Lamin A, suggesting their roles in transcription and splicing. Clinical analysis revealed that high expressions of MMP3 and LMNA were significantly seen in head and neck squamous cell carcinoma (HNSC) than in the other 16 cancer types, and predicted poor prognosis of patients suffering from HNSC, pancreatic, rectum and lung adenocarcinomas at specific stages. Immunohistochemistry revealed that nuclear MMP-3 and cleaved Lamin were significantly higher expressed in stage IV metastatic HNSC cases than in stage I non-metastatic cases. Taken together, MMP3-cleavage of Lamin A induces nuclear deformity, nucleophagy, and their autophagic co-secretion with EVs in metastatic cancer. Also, high expression of MMP-3 and secretion of Lamin A can predict poor prognosis in multiple cancer types at specific stages. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0006-291X 752 2025 Discovery of myeloid zinc finger (MZF) 1 nuclear bodies 151481 EN Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Myeloid zinc finger 1 (MZF1) is a multifaceted transcription factor that can act either as a transcriptional activator or a gene repressor. We examined its production of nuclear bodies (NBs) and subcellular localization. Proteomic and protein–protein interaction analysis were used to identify its cofactors and interactions. These revealed the presence of MZF1-NBs (intranuclear oligomers containing MZF1). MZF-NBs are similar to some other nuclear bodies, notably promyelocytic leukemia (PML) -NBs in terms of size and morphology. However the two structures appear to be different. MZF-NBs and PML-NBs were found to associate in the nucleus. Both MZF1 and PML are SUMO1-SUMOylated in PC-3 cells. Sumoylated MZF1 can interact with proteins containing SUMO-interaction motifs (SIM) through SUMO-SIM interaction. Interactome analysis revealed that its NBs participate in the stress response (TPR and UBAP2L), protein folding (CALR and ANKRD40), transcription, post-translational modification (TRIM33, ACOT7, CAMK2D, and CAMK2G), and RNA binding (ALURBP and CPSF5). No potential conflict of interest relevant to this article was reported.

���R��w�� Acta Medica Okayama 0030-1558 136 1 2024 �G�N�\�\�[�� 33 36 EN Takanori Eguchi Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1368-8375 142 2023 EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma 106433 EN Koki Umemori Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoya Nakamura Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tatsuo Ogawa Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kunihiro Yoshida Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideka Kanemoto Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kohei Sato Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kyoichi Obata Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shoji Ryumon Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirokazu Yutori Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoki Katase Department of Oral Pathology, Graduate School of Biomedical Sciences, Nagasaki University Tatsuo Okui Department of Oral and Maxillofacial Surgery, Shimane University Faculty of Medicine Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Soichiro Ibaragi Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Objectives: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance.<br> Materials and methods: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS).<br> Results: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner.<br> Conclusion: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 24 6 2023 Stress-Inducible SCAND Factors Suppress the Stress Response and Are Biomarkers for Enhanced Prognosis in Cancers 5168 EN Mona Sheta Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kunihiro Yoshida Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideka Kanemoto Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Takanori Eguchi Department of Dental Pharmacology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The cell stress response is an essential system present in every cell for responding and adapting to environmental stimulations. A major program for stress response is the heat shock factor (HSF)-heat shock protein (HSP) system that maintains proteostasis in cells and promotes cancer progression. However, less is known about how the cell stress response is regulated by alternative transcription factors. Here, we show that the SCAN domain (SCAND)-containing transcription factors (SCAN-TFs) are involved in repressing the stress response in cancer. SCAND1 and SCAND2 are SCAND-only proteins that can hetero-oligomerize with SCAN-zinc finger transcription factors, such as MZF1(ZSCAN6), for accessing DNA and transcriptionally co-repressing target genes. We found that heat stress induced the expression of SCAND1, SCAND2, and MZF1 bound to HSP90 gene promoter regions in prostate cancer cells. Moreover, heat stress switched the transcript variants' expression from long noncoding RNA (lncRNA-SCAND2P) to protein-coding mRNA of SCAND2, potentially by regulating alternative splicing. High expression of HSP90AA1 correlated with poorer prognoses in several cancer types, although SCAND1 and MZF1 blocked the heat shock responsiveness of HSP90AA1 in prostate cancer cells. Consistent with this, gene expression of SCAND2, SCAND1, and MZF1 was negatively correlated with HSP90 gene expression in prostate adenocarcinoma. By searching databases of patient-derived tumor samples, we found that MZF1 and SCAND2 RNA were more highly expressed in normal tissues than in tumor tissues in several cancer types. Of note, high RNA expression of SCAND2, SCAND1, and MZF1 correlated with enhanced prognoses of pancreatic cancer and head and neck cancers. Additionally, high expression of SCAND2 RNA was correlated with better prognoses of lung adenocarcinoma and sarcoma. These data suggest that the stress-inducible SCAN-TFs can function as a feedback system, suppressing excessive stress response and inhibiting cancers. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2079-7737 12 1 2023 Extracellular Vesicles: New Classification and Tumor Immunosuppression 110 EN Mona Sheta Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eman A. Taha Department of Biochemistry, Faculty of Science, Ain Shams University Yanyin Lu Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Simple Summary Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules and deliver them to recipient cells. Classical EVs are exosomes, microvesicles, and apoptotic bodies. This review classifies classical and additional EV types, including autophagic EVs, matrix vesicles, and stressed EVs. Of note, matrix vesicles are key components interacting with extracellular matrices (ECM) in the tumor microenvironment. We also review how EVs are involved in the communication between cancer cells and tumor-associated cells (TAC), leading to establishing immunosuppressive and chemoresistant microenvironments. These include cancer-associated fibroblasts (CAF), mesenchymal stem cells (MSC), blood endothelial cells (BEC), lymph endothelial cells (LEC), and immune cells, such as tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), dendritic cells, natural killer cells, killer T cells, and immunosuppressive cells, such as regulatory T cells and myeloid-derived suppressor cells (MDSC). Exosomal long noncoding RNA (lncRNA), microRNA, circular RNA, piRNA, mRNA, and proteins are crucial in communication between cancer cells and TACs for establishing cold tumors. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles carrying various types of molecules. These EV cargoes are often used as pathophysiological biomarkers and delivered to recipient cells whose fates are often altered in local and distant tissues. Classical EVs are exosomes, microvesicles, and apoptotic bodies, while recent studies discovered autophagic EVs, stressed EVs, and matrix vesicles. Here, we classify classical and new EVs and non-EV nanoparticles. We also review EVs-mediated intercellular communication between cancer cells and various types of tumor-associated cells, such as cancer-associated fibroblasts, adipocytes, blood vessels, lymphatic vessels, and immune cells. Of note, cancer EVs play crucial roles in immunosuppression, immune evasion, and immunotherapy resistance. Thus, cancer EVs change hot tumors into cold ones. Moreover, cancer EVs affect nonimmune cells to promote cellular transformation, including epithelial-to-mesenchymal transition (EMT), chemoresistance, tumor matrix production, destruction of biological barriers, angiogenesis, lymphangiogenesis, and metastatic niche formation. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 11 24 2022 SCAND1 Reverses Epithelial-to-Mesenchymal Transition (EMT) and Suppresses Prostate Cancer Growth and Migration 3993 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eva Csizmadia Division of Surgical Sciences, Department of Surgery, Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School Hotaka Kawai Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mona Sheta Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kunihiro Yoshida Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Thomas L. Prince Ranok Therapeutics Barbara Wegiel Division of Surgical Sciences, Department of Surgery, Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School 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 carcinomas depend on EMT activation, partial EMT, or 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 prostate epithelial transcription factor SCAND1 can reverse the cancer cell mesenchymal and hybrid E/M phenotypes to a more epithelial, less invasive status and inhibit their proliferation and migration in DU-145 prostate cancer cells. SCAND1 is a SCAN domain-containing protein and hetero-oligomerizes with SCAN-zinc finger transcription factors, such as MZF1, for accessing DNA and the transcriptional co-repression of target genes. We found that SCAND1 expression correlated with maintaining epithelial features, whereas the loss of SCAND1 was associated with mesenchymal phenotypes of tumor cells. SCAND1 and MZF1 were mutually inducible and coordinately included in chromatin with hetero-chromatin protein HP1 gamma. The overexpression of SCAND1 reversed hybrid E/M status into an epithelial phenotype with E-cadherin and beta-catenin relocation. Consistently, the co-expression analysis in TCGA PanCancer Atlas revealed that SCAND1 and MZF1 expression was negatively correlated with EMT driver genes, including CTNNB1, ZEB1, ZEB2 and TGFBRs, in prostate adenocarcinoma specimens. In addition, SCAND1 overexpression suppressed tumor cell proliferation by reducing the MAP3K-MEK-ERK signaling pathway. Of note, in a mouse tumor xenograft model, SCAND1 overexpression significantly reduced Ki-67(+) and Vimentin(+) tumor cells and inhibited migration and lymph node metastasis of prostate cancer. Kaplan-Meier analysis showed high expression of SCAND1 and MZF1 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 SCAND1 induces expression and coordinated heterochromatin-binding of MZF1 to reverse the hybrid E/M status into an epithelial phenotype and, inhibits tumor cell proliferation, migration, and metastasis, potentially by repressing the gene expression of EMT drivers and the MAP3K-MEK-ERK signaling pathway. No potential conflict of interest relevant to this article was reported.

American Society for Clinical Investigation Acta Medica Okayama 2379-3708 7 1 2022 Resident stroma-secreted chemokine CCL2 governs myeloid-derived suppressor cells in the tumor microenvironment e148960 EN May Wathone Oo Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kiyofumi Takabatake Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shuta Tomida Center for Comprehensive Genomic Medicine, Okayama University Hospital Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Qiusheng Shan Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Saori Yoshida Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Haruka Omori Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Shintaro Sukegawa Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keisuke Nakano Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akira Sasaki Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Accumulating evidence has shown that cancer stroma and BM-derived cells (BMDCs) in the tumor microenvironment (TME) play vital roles in tumor progression. However, the mechanism by which oral cancer stroma recruits any particular subset of BMDCs remains largely unknown. Here, we sought to identify the subset of BMDCs that is recruited by cancer stroma. We established a sequential transplantation model in BALB/c nude mice, including (a) BM transplantation of GFP-expressing cells and (b) coxenografting of patient-derived stroma (PDS; 2 cases, designated PDS1 and PDS2) with oral cancer cells (HSC-2). As controls, xenografting was performed with HSC-2 alone or in combination with normal human dermal fibroblasts (HDF). PDS1, PDS2, and HDF all promoted BMDC migration in vitro and recruitment in vivo. Multicolor immunofluorescence revealed that the PDS coxenografts recruited Arginase-1(+)CD11b(+)GR1(+)GFP(+) cells, which are myeloid-derived suppressor cells (MDSCs), to the TME, whereas the HDF coxenograft did not. Screening using microarrays revealed that PDS1 and PDS2 expressed CCL2 mRNA (encoding C-C motif chemokine ligand 2) at higher levels than did HDF. Indeed, PDS xenografts contained significantly higher proportions of CCL2(+) stromal cells and CCR2(+)Arginase-1(+)CD11b(+)GR1(+) MDSCs (as receiver cells) than the HDF coxenograft. Consistently, a CCL2 synthesis inhibitor and a CCR2 antagonist significantly inhibited the PDS-driven migration of BM cells in vitro. Furthermore, i.p. injection of the CCR2 antagonist to the PDS xenograft models significantly reduced the CCR2(+)Arginase-1(+)CD11b(+)GR1(+) MDSC infiltration to the TME. In conclusion, oral cancer stroma-secreted CCL2 is a key signal for recruiting CCR2(+) MDSCs from BM to the TME. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 1044-579X 86 1 2022 Cancer extracellular vesicles, tumoroid models, and tumor microenvironment 112 126 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mona Sheta Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masanori Fujii Department of Allergy and Respiratory Medicine, Okayama University Hospital Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Cancer extracellular vesicles (EVs), or exosomes, promote tumor progression through enhancing tumor growth, initiating epithelial-to-mesenchymal transition, remodeling the tumor microenvironment, and preparing metastatic niches. Three-dimensionally (3D) cultured tumoroids / spheroids aim to reproduce some aspects of tumor behavior in vitro and show increased cancer stem cell properties. These properties are transferred to their EVs that promote tumor growth. Moreover, recent tumoroid models can be furnished with aspects of the tumor microenvironment, such as vasculature, hypoxia, and extracellular matrix. This review summarizes tumor tissue culture and engineering platforms compatible with EV research. For example, the combination experiments of 3D-tumoroids and EVs have revealed multifunctional proteins loaded in EVs, such as metalloproteinases and heat shock proteins. EVs or exosomes are able to transfer their cargo molecules to recipient cells, whose fates are often largely altered. In addition, the review summarizes approaches to EV labeling technology using fluorescence and luciferase, useful for studies on EV-mediated intercellular communication, biodistribution, and metastatic niche formation. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 10 6 2021 Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method 1328 EN Yanyin Lu Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chiharu Sogawa Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eman A. Taha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Manh Tien Tran Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Nara Research Program for Undergraduate Students, Okayama University Dental School Penggong Wei Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shiro Fukuoka Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuya Miyawaki Department of Dental Anesthesiology and Special Care Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kuniaki Okamoto Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500-3000 nm) reminiscent of apoptosomes, (ii) EVs (100-500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80-300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20-200 nm) containing unidentified vesicles/particles, and (v) EVs (10-70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producing a GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1574-7891 12 8 2018 Genotoxic stress induces Sca�]1�]expressing metastatic mammary cancer cells 1249 1263 EN Jianlin Gong Department of Medicine, Boston University Medical Center Benjamin J. Lang Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Desheng Weng Department of Medicine, Boston University Medical Center Takanori Eguchi Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Ayesha Murshid Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Thiago J. Borges Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Sachin Doshi Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Baizheng Song Department of Medicine, Boston University Medical Center Mary A. Stevenson Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School We describe a cell damage�]induced phenotype in mammary carcinoma cells involving acquisition of enhanced migratory and metastatic properties. Induction of this state by radiation required increased activity of the Ptgs2 gene product cyclooxygenase 2 (Cox2), secretion of its bioactive lipid product prostaglandin E2 (PGE2), and the activity of the PGE2 receptor EP4. Although largely transient, decaying to low levels in a few days to a week, this phenotype was cumulative with damage and levels of cell markers Sca�]1 and ALDH1 increased with treatment dose. The Sca�]1+, metastatic phenotype was inhibited by both Cox2 inhibitors and PGE2 receptor antagonists, suggesting novel approaches to radiosensitization. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2077-0383 9 11 2020 Organoids and Liquid Biopsy in Oral Cancer Research 3701 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 10 2 2021 Gel-Free 3D Tumoroids with Stem Cell Properties Modeling Drug Resistance to Cisplatin and Imatinib in Metastatic Colorectal Cancer 344 EN Chiharu Sogawa Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuri Namba Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuka Okusha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eriko Aoyama Advanced Research Center for Oral and Craniofacial Sciences (ARCOCS), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazumi Ohyama Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kuniaki Okamoto Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 ��M), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 ��M) and Imatinib (10 ��M) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance. No potential conflict of interest relevant to this article was reported.

Taylor & Francis Acta Medica Okayama 2001-3078 9 1 2020 Triple knockdown of CDC37, HSP90�]alpha and HSP90�]beta diminishes extracellular vesicles�]driven malignancy events and macrophage M2 polarization in oral cancer 1769373 EN Kisho Ono Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman A. Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yanyin Lu Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University May Wathone Oo Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Soichiro Ibaragi Department of Oral and Maxillofacial Surgery, Okayama University Hospital Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Akira Sasaki Department of Oral and Maxillofacial Surgery, Okayama University Hospital Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones – CDC37, HSP90�� and HSP90�� play key roles in cancer progression including epithelial�]mesenchymal transition (EMT), although their contribution to EVs�]mediated cell–cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer�]derived EVs (MEV) were enriched with HSP90�� HSP90�� and cancer�]initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90��/�� reversed these MEV�]driven malignancy events. In metastatic oral cancer patient�]derived tumours, HSP90�� was significantly accumulated in infiltrating tumour�]associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90�]enriched MEV�]induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA�]mediated knockdown of the chaperone trio (CDC37/HSP90��/HSP90��) could potentially be a novel therapeutic strategy to attenuate several EV�]driven malignancy events in the tumour microenvironment. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 21 24 2020 The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors 9352 EN Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yunxia Feng Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masatoshi Morimatsu Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Penggong Wei Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoko Kadowaki Department of Frontier Oral Science, Graduate School of Biomedical Sciences, Nagasaki University Eiko Sakai Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Hirohiko Okamura Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiji Naruse Department of Cardiovascular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takayuki Tsukuba Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 9 11 2020 Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors 2384 EN Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Mami Itagaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tatsuo Okui Department of Oral and Maxillofacial Surgery and Biopathology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tomoko Kadowaki Department of Frontier Life Science, Graduate School of Biomedical Sciences, Nagasaki University Eiko Sakai Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Takayuki Tsukuba Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-kappa B ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-kappa B (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes-late endosomes-lysosomes in osteoclasts. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 11 6 2019 MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer 792 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Thomas L Prince Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Benjamin J Lang Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family-MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2079-7737 9 3 2020 A Novel Model of Cancer Drug Resistance: Oncosomal Release of Cytotoxic and Antibody-Based Drugs 47 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman Ahmed Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Hospital Extracellular vesicles (EVs), such as exosomes or oncosomes, often carry oncogenic molecules derived from tumor cells. In addition, accumulating evidence indicates that tumor cells can eject anti-cancer drugs such as chemotherapeutics and targeted drugs within EVs, a novel mechanism of drug resistance. The EV-releasing drug resistance phenotype is often coupled with cellular dedifferentiation and transformation in cells undergoing epithelial-mesenchymal transition (EMT), and the adoption of a cancer stem cell phenotype. The release of EVs is also involved in immunosuppression. Herein, we address different aspects by which EVs modulate the tumor microenvironment to become resistant to anticancer and antibody-based drugs, as well as the concept of the resistance-associated secretory phenotype (RASP). No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 5 2020 Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles 1260 EN Eman A. Taha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Chiharu Sogawa Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuka Okusha Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Wathone Oo Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Abdellatif Elseoudi Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yanyin Lu Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ayano Satoh Department of Medical Bioengineering, Okayama University Graduate School of Natural Science and Technology Kuniaki Okamoto Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 4 2020 Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer 881 EN Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manh T. Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kaya Yoshida Department of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima University Graduate School Mami Itagaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman A. Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Hospital Eriko Aoyama Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirohiko Okamura Department of Oral Morphology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 9 3 2020 Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells 755 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Hospital Masaki Matsumoto Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Tran Manh Tien Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Benjamin J. Lang Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90 alpha was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90 alpha, and HSP90 beta was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 2 2020 Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3 523 EN Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Masayuki Ishige On-Chip Biotechnologies, Co., Ltd. Kilian Trin Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Eman Ahmed Taha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yanyin Lu Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hotaka Kawai Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Norio Sogawa Department of Dental Pharmacology, Matsumoto Dental University Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-kappa B, and beta-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 1019-6439 54 1 2018 Nicotine promotes lymph node metastasis and cetuximab resistance in head and neck squamous cell carcinoma. 283 294 EN Rieko Shimizu Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Soichiro Ibaragi Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Daisuke Kuwajima Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Shinichi Kodama Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Takashi Nishioka Department of Oral Diagnosis, Tohoku University Graduate School of Dentistry Tatsuo Okui Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kyoichi Obata Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kiyofumi Takabatake Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hotaka Kawai Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Kuniaki Okamoto Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Akira Sasaki Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Epidermal growth factor (EGF) is overexpressed in many cancers and is associated with worse prognosis. EGF binds to its cell surface receptor (EGFR), which induces EGFR phosphorylation. Phosphorylated EGFR (p‑EGFR) is translocated into the nucleus, which increases cancer cell activity. Nicotine, which is one of the main components of tobacco, is absorbed through pulmonary alveoli and mucosal epithelia in the head and neck region by smoking and moves into the blood. Nicotine in blood binds to nicotinic acetylcholine receptor (nAChR) in the central nervous system and serves a crucial role in tobacco addiction. Although nAChR localization is thought to be limited in the nervous system, nAChR is present in a wide variety of non‑neuronal cells, including cancer cells. Recent studies suggest that nicotine contributes to the metastasis and resistance to anti‑cancer drugs of various cancer cells. However, it remains unknown whether head and neck squamous cell carcinoma (HNSCC) cells can utilize nicotine‑nAChR signaling to metastasize and acquire resistance to anti‑cancer drugs, even though the mucosal epithelia of the head and neck region are the primary sites of exposure to tobacco smoke. To the best of our knowledge, the present study is the first to demonstrate the role of nicotine in metastasis and anti‑EGFR‑therapy resistance of HNSCC. The present findings demonstrated that nicotine increased proliferation, migration, invasion, p‑EGFR nuclear translocation and protein kinase B (Akt) phosphorylation in HNSCC cells. It was also demonstrated that nicotine restored cetuximab‑inhibited proliferation, migration and invasion of HNSCC cells. Finally, an in vivo experiment revealed that nicotine increased lymph node metastasis of xenografted tumors, whereas an nAChR inhibitor suppressed lymph node metastasis and p‑EGFR nuclear localization of xenografted tumors. Taken together, these results demonstrated that nicotine induced nuclear accumulation of p‑EGFR, and activation of Akt signaling. These signaling pathways elevated the activities of HNSCC cells, causing lymph node metastasis and serving a role in cetuximab resistance. No potential conflict of interest relevant to this article was reported.

Molecular Diversity Preservation International Acta Medica Okayama 20726694 11 6 2019 MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer E792 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Thomas L. Prince Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Benjamin J. Lang Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family-MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1. No potential conflict of interest relevant to this article was reported.

Mary Ann Liebert Acta Medica Okayama 19373341 25 19-20 2019 A reporter system evaluates tumorigenesis, metastasis, ��-catenin/MMP regulation, and druggability 1413 1425 EN Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kazumi Ohyama Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Motoharu Iizuka Research program for undergraduate students, Okayama University Dental School Ryu Kawasaki Research program for undergraduate students, Okayama University Dental School Yusaku Hamada Research program for undergraduate students, Okayama University Dental School Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norio Sogawa Department of Dental Pharmacology, Matsumoto Dental University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken-ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high-expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. Additionally, a new strategy for anti-cancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and Mmp9 promoter and (ii) to examine pharmacological actions of anti-cancer medications using this reporter system. High expression and genetic amplification of MMP9 were found in colon cancer cases. We found that proximal promoter sequences of MMP9 in murine and human contained conserved binding sites for transcription factors ��-catenin/TCF/LEF, glucocorticoid receptor (GR), and NF-��B. The murine Mmp9 promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by ��-catenin signaling stimulator lithium chloride (LiCl). The Mmp9 promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in allogeneic/syngenic transplantation experiments. We also demonstrated pharmacological actions as follows. ids Dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the Mmp9 promoter but did not inhibit tumorigenesis. On the other hand, an antimetabolite 5-fluorouracil, a golden standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit Mmp9 promoter activity. Notably, anti-malaria medication artesunate inhibited both tumorigenesis and the Mmp9 promoter in vitro, potentially through inhibition of ��-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as ��-catenin/MMP9 axis, and druggability. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 20726694 11 6 2019 MZF1 and SCAND1 Reciprocally Regulate CDC37 Gene Expression in Prostate Cancer 792 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Thomas L. Prince Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Manh Tien Tran Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Benjamin J. Lang Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K. Calderwood Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Cell division control 37 (CDC37) increases the stability of heat shock protein 90 (HSP90) client proteins and is thus essential for numerous intracellular oncogenic signaling pathways, playing a key role in prostate oncogenesis. Notably, elevated expression of CDC37 was found in prostate cancer cells, although the regulatory mechanisms through which CDC37 expression becomes increased are unknown. Here we show both positive and negative regulation of CDC37 gene transcription by two members of the SREZBP-CTfin51-AW1-Number 18 cDNA (SCAN) transcription factor family�\MZF1 and SCAND1, respectively. Consensus DNA-binding motifs for myeloid zinc finger 1 (MZF1/ZSCAN6) were abundant in the CDC37 promoter region. MZF1 became bound to these regulatory sites and trans-activated the CDC37 gene whereas MZF1 depletion decreased CDC37 transcription and reduced the tumorigenesis of prostate cancer cells. On the other hand, SCAND1, a zinc fingerless SCAN box protein that potentially inhibits MZF1, accumulated at MZF1-binding sites in the CDC37 gene, negatively regulated the CDC37 gene and inhibited tumorigenesis. SCAND1 was abundantly expressed in normal prostate cells but was reduced in prostate cancer cells, suggesting a potential tumor suppressor role of SCAND1 in prostate cancer. These findings indicate that CDC37, a crucial protein in prostate cancer progression, is regulated reciprocally by MZF1 and SCAND1. No potential conflict of interest relevant to this article was reported.

SAGE-Hindawi Access to Research Acta Medica Okayama 1687-966X 2016 2016 Cellular Reprogramming Using Defined Factors and MicroRNAs 7530942 EN Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuo Kuboki Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0730-2312 116 10 2015 Role and Regulation of Myeloid Zinc Finger Protein 1 in Cancer 2146 2154 EN Taka Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Thomas Prince Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute Barbara Wegiel Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Myeloid zinc finger 1 (MZF1) belongs to the SCAN-Zinc Finger (SCAN-ZF) transcription factor family that has recently been implicated in a number of types of cancer. Although the initial studies concentrated on the role of MZF1 in myeloid differentiation and leukemia, the factor now appears to be involved in the etiology of major solid tumors such as lung, cervical, breast, and colorectal cancer. Here we discuss the regulation of MZF1 that mediated its recruitment and activation in cancer, concentrating on posttranslational modification by phosphorylation, and sumoylation, formation of promyelocytic leukemia nuclear bodies and its association with co-activators and co-repressors. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 1097-4644 119 9 2018 The intranuclear PEX domain of MMP involves proliferation, migration, and metastasis of aggressive adenocarcinoma cells 7363 7376 EN Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tatsuo Okui Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keisuke Nakano Advanced Research Center for Oral and Craniofacial Sciences, Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken�]Ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells. No potential conflict of interest relevant to this article was reported.

American Society for Microbiology Acta Medica Okayama 0270-7306 28 7 2008 Novel transcription-factor-like function of human matrix metalloproteinase 3 regulating the CTGF/CCN2 gene 2391 2413 EN Takanori Eguchi Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazumi Kawata Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiki Mukudai Bio-Dental Research Center, Okayama University Dental School Junji Uehara Department of Oral & Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshihiro Ohgawara Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Soichiro Ibaragi Department of Oral & Maxillofacial Surgery & Biopathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Sasaki Department of Oral & Maxillofacial Surgery & Biopathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Takuo Kuboki Department of Oral & Maxillofacial Rehabilitation, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaharu Takigawa Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences �@Matrix metalloproteinase 3 (MMP3) is well known as a secretory endopeptidase that degrades extracellular matrices. Recent reports indicated the presence of MMPs in the nucleus (A. J. Kwon et al., FASEB J. 18:690-692, 2004); however, its function has not been well investigated. Here, we report a novel function of human nuclear MMP3 as a trans regulator of connective tissue growth factor (CCN2/CTGF). Initially, we cloned MMP3 cDNA as a DNA-binding factor for the CCN2/CTGF gene. An interaction between MMP3 and transcription enhancer dominant in chondrocytes (TRENDIC) in the CCN2/CTGF promoter was confirmed by a gel shift assay and chromatin immunoprecipitation. The CCN2/CTGF promoter was activated by overexpressed MMP3, whereas a TRENDIC mutant promoter lost the response. Also, the knocking down of MMP3 suppressed CCN2/CTGF expression. By cytochemical and histochemical analyses, MMP3 was detected in the nuclei of chondrocytic cells in culture and also in the nuclei of normal and osteoarthritic chondrocytes in vivo. The nuclear translocation of externally added recombinant MMP3 and six putative nuclear localization signals in MMP3 also were shown. Furthermore, we determined that heterochromatin protein gamma coordinately regulates CCN2/CTGF by interacting with MMP3. The involvement of this novel role of MMP3 in the development, tissue remodeling, and pathology of arthritic diseases through CCN2/CTGF regulation thus is suggested. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 07302312 118 1 2016 Intracellular MMP3 Promotes HSP Gene Expression in Collaboration With Chromobox Proteins 43 51 EN Takanori Eguchi Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Masaharu Takigawa Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken�]ichi Kozaki Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Matrix metalloproteinases (MMPs) are crucial factors in tumor progression, inflammatory/immune responses and tissue development/regeneration. Of note, it has been known that MMPs promote genome instability, epithelial-mesenchymal transition, invasion, and metastasis in tumor progression. We previously reported that human MMP3 could translocate into cellular nuclei and control transcription in human chondrosarcoma-derived cells and in articular cartilage (Eguchi et al. [2008] Mol Cell Biol 28(7):2391-2413); however, further transcriptional target genes and cofactors of intranuclear MMP3 have not been uncovered. In this paper, we used transcriptomics analysis in order to examine novel transcriptional target genes regulated by intracellular MMP3. We found that mRNA levels of HSP family members (HSP70B', HSP72, HSP40/DNAJ, and HSP20/CRYAB) are upregulated by the intracellular MMP3 overload. Bioinformatic analysis predicted several transcription factors that possibly interact with MMP3. Among these factors, heat shock factor 1 (HSF1) cooperated with the MMP3 to activate the HSP70B' gene promoter in reporter gene assays, while a dominant negative HSF1 blocked the role for MMP3 in the trans-activation. The hemopexin-like repeat (PEX) domain of the human MMP3 was essential for transcriptional induction of the HSP70B' gene. In addition, chromobox proteins CBX5/HP1�� and CBX3/HP1�� cooperated with the PEX domain in induction of HSP70B' mRNA. Taken together, this study newly clarified that intracellular MMP3 cooperate with CBXs/HP1s in transcriptional promotion of HSP genes. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 07302312 119 9 2018 HSP�]enriched properties of extracellular vesicles involve survival of metastatic oral cancer cells 7350 7362 EN Kisho Ono Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Junya Futagawa Department of Biomedical Solution Center, Mitsui Knowledge Industry Tomonari Kasai Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Masaharu Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Akira Sasaki Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Ken�]ichi Kozaki Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Cancer cells often secrete extracellular vesicles (EVs) that carry heat shock proteins (HSPs) with roles in tumor progression. Oral squamous cell carcinoma (OSCC) belongs to head and neck cancers (HNC) whose lymph-node-metastases often lead to poor prognosis. We have examined the EV proteome of OSCC cells and found abundant secretion of HSP90-enriched EVs in lymph-node-metastatic OSCC cells. Double knockdown of HSP90�� and HSP90��, using small interfering RNA significantly reduced the survival of the metastatic OSCC cells, although single knockdown of each HSP90 was ineffective. Elevated expression of these HSP90 family members was found to correlate with poor prognosis of HNC cases. Thus, elevated HSP90 levels in secreted vesicles are potential prognostic biomarkers and therapeutic targets in metastatic OSCC. No potential conflict of interest relevant to this article was reported.