Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 2025 S100A8/A9-MCAM signaling promotes gastric cancer cell progression via ERK-c-Jun activation EN Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Xu Yang Department of Pathology, The First Affiliated Hospital, Zhejiang University School of Medicine Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Bo Pan The First Affiliated Hospital, Zhejiang University School of Medicine Fangping Wu School of Pharmaceutical Sciences, Zhejiang Chinese Medical University Xu Zhang Department of Urology, The First Affiliated Hospital, Zhejiang University School of Medicine Kazumi Sagayama Faculties of Educational and Research Management Field, Okayama University Bei Sun Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences S100 protein family members S100A8 and S100A9 function primarily as a heterodimer complex (S100A8/A9) in vivo. This complex has been implicated in various cancers, including gastric cancer (GC). Recent studies suggest that these proteins play significant roles in tumor progression, inflammation, and metastasis. However, the exact mechanisms by which S100A8/A9 contributes to GC pathogenesis remain unclear. This study investigates the role of S100A8/A9 and its receptor in GC. Immunohistochemical analysis was performed on GC tissue samples to assess the expression of the S100A8/A9 receptor melanoma cell adhesion molecule (MCAM). In vitro transwell migration and invasion assays were used to evaluate the motility and invasiveness of GC cells. Cell proliferation was assessed using a growth assay, and Western blotting (WB) was employed to examine downstream signaling pathways, including ERK and the transcription factor c-Jun, in response to S100A8/A9–MCAM interaction. S100A8/A9 stimulation enhanced both proliferation and migration through MCAM binding in GC cell lines. These cellular events were accompanied by ERK activation and c-Jun induction. Downregulation of MCAM suppressed both ERK phosphorylation and c-Jun expression, highlighting the importance of the S100A8/A9‒MCAM‒ERK‒c-Jun axis in promoting GC progression. These findings indicate that S100A8/A9 contributes to GC progression via MCAM, which activates the ERK‒c-Jun pathway. The S100A8/A9‒signaling axis may represent a novel therapeutic target in GC. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells 1215 1227 EN Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Tetta Takahashi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Toshiki Ochi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine I Made Winarsa Ruma Faculty of Medicine, Udayana University I Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Kazumi Sagayama Organization for Research and Innovation Strategy, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector’s shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy. No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 2076-3921 14 7 2025 Carnosol, a Rosemary Ingredient Discovered in a Screen for Inhibitors of SARM1-NAD+ Cleavage Activity, Ameliorates Symptoms of Peripheral Neuropathy 808 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuki Ogawa Tama Biochemical Co., Ltd. Yu Yasui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoji Wada Tama Biochemical Co., Ltd. Hiromichi Nakamura Tama Biochemical Co., Ltd. Masahiro Nishibori Department of Translational Research and Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+) hydrolase involved in axonal degeneration and neuronal cell death. SARM1 plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents the degeneration; as a result, SARM1 has been attracting attention as a potent therapeutic target. In recent years, the development of several SARM1 inhibitors derived from synthetic chemical compounds has been reported; however, no dietary ingredients with SARM1 inhibitory activity have been identified. Therefore, we here focused on dietary ingredients and found that carnosol, an antioxidant contained in rosemary, inhibits the NAD+-cleavage activity of SARM1. Purified carnosol inhibited the enzymatic activity of SARM1 and suppressed neurite degeneration and cell death induced by the anti-cancer medicine vincristine (VCR). Carnosol also inhibited VCR-induced hyperalgesia symptoms, suppressed the loss of intra-epidermal nerve fibers in vivo, and reduced the blood fluid level of phosphorylated neurofilament-H caused by an axonal degeneration event. These results indicate that carnosol has a neuroprotective effect via SARM1 inhibition in addition to its previously known antioxidant effect via NF-E2-related factor 2 and thus suppresses neurotoxin-induced peripheral neuropathy. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 15 1 2025 Plasma S100A8/A9 level predicts response to immune checkpoint inhibitors in patients with advanced non-small cell lung cancer 2577 EN Tadahiro Kuribayashi Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kiichiro Ninomiya Department of Allergy and Respiratory Medicine, Okayama University Hospital Go Makimoto Department of Allergy and Respiratory Medicine, Okayama University Hospital Toshio Kubo Department of Allergy and Respiratory Medicine, Okayama University Hospital Kammei Rai Department of Allergy and Respiratory Medicine, Okayama University Hospital Eiki Ichihara Center for Clinical Oncology, Okayama University Hospital Katsuyuki Hotta Department of Allergy and Respiratory Medicine, Okayama University Hospital Masahiro Tabata Department of Allergy and Respiratory Medicine, Okayama University Hospital Yoshinobu Maeda Department of Hematology, Oncology and Respiratory Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Katsuyuki Kiura Department of Allergy and Respiratory Medicine, Okayama University Hospital Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kadoaki Ohashi Department of Allergy and Respiratory Medicine, Okayama University Hospital Blood-based predictive markers for the efficacy of immune checkpoint inhibitors (ICIs) have not yet been established. We investigated the association of the plasma level of S100A8/A9 with the efficacy of immunotherapy. We evaluated patients with unresectable stage III/IV or recurrent non-small cell lung cancer (NSCLC) who were treated with ICIs at Okayama University Hospital. The pre-treatment plasma levels of S100A8/A9 were analyzed. Eighty-one eligible patients were included (median age, 69 years). Sixty-two patients were men, 54 had adenocarcinoma, 74 had performance status (PS) 0–1, and 47 received ICIs as first-line treatment. The median time to treatment failure (TTF) for ICIs was 5.7 months, and the median overall survival (OS) was 19.6 months. The TTF and OS were worse in patients with high plasma S100A8/A9 levels (≥ 2.475 µg/mL) (median TTF: 4.3 vs. 8.5 months, p = 0.009; median OS: 15.4 vs. 38.0 months, p = 0.001). Multivariate analysis revealed that PS ≥ 2, liver metastasis, and high plasma S100A8/A9 levels were significantly associated with short TTF and OS. In conclusion, plasma S100A8/A9 level may have a limited effect on ICI therapy for NSCLC. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 1071-2690 60 10 2024 S100A11 is involved in the progression of colorectal cancer through the desmosome-catenin-TCF signaling pathway 1138 1149 EN Jin Zhou Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Naoko Mizuta Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Atsuko Yamakawa Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Compiling evidence has indicated that S100A11 expression at high levels is closely associated with various cancer species. Consistent with the results reported elsewhere, we have also revealed that S100A11 is highly expressed in squamous cell carcinoma, mesothelioma, and pancreatic cancers and plays a crucial role in cancer progression when secreted into extracellular fluid. Those studies are all focused on the extracellular role of S100A11. However, most of S100A11 is still present within cancer cells, although the intracellular role of S100A11 in cancer cells has not been fully elucidated. Thus, we aimed to investigate S100A11 functions within cancer cells, primarily focusing on colorectal cancer cells, whose S100A11 is abundantly present in cells and still poorly studied cancer for the protein. Our efforts revealed that overexpression of S100A11 promotes proliferation and migration, and downregulation inversely dampens those cancer behaviors. To clarify how intracellular S100A11 aids cancer cell activation, we tried to identify S100A11 binding proteins, resulting in novel binding partners in the inner membrane, many of which are desmosome proteins. Our molecular approach defined that S100A11 regulates the expression level of DSG1, a component protein of desmosome, by which S100A11 activates the TCF pathway via promoting nuclear translocation of γ-catenin from the desmosome. The identified new pathway greatly helps to comprehend S100A11’s nature in colorectal cancers and others. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-β1-NF-κB-MMP9 axis 1371307 EN Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Kazumi Ninomiya Faculty of Science and Technology, Division of Molecular Science, Gunma University Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-beta 1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-beta 1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-kappa B (NF-kappa B). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-beta activated kinase 1 (TAK1) were required for the activation of NF-kappa B through I kappa beta kinase kinase (IKK alpha/beta) phosphorylation. Conclusion Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-beta 1-TRAF4-TAK1-IKK alpha/beta-I kappa beta alpha-NF-kappa B-MMP9, is crucial for TNBC cell invasiveness. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0946-2716 101 4 2023 Novel extracellular role of REIC/Dkk-3 protein in PD-L1 regulation in cancer cells 431 447 EN Yuma Gohara Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Nahoko Tomonobu Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Léna Audebert Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Youyi Chen Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ni Luh Gede Yoni Komalasari Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Fan Jiang Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Chikako Yoshizawa Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitoshi Murata Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Ken-ichi Yamamoto Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Masami Watanabe Department of Urology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Masakiyo Sakaguchi Department of Cell Biology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine The adenovirus-REIC/Dkk-3 expression vector (Ad-REIC) has been the focus of numerous clinical studies due to its potential for the quenching of cancers. The cancer-suppressing mechanisms of the REIC/DKK-3 gene depend on multiple pathways that exert both direct and indirect effects on cancers. The direct effect is triggered by REIC/Dkk-3-mediated ER stress that causes cancer-selective apoptosis, and the indirect effect can be classified in two ways: (i) induction, by Ad-REIC-mis-infected cancer-associated fibroblasts, of the production of IL-7, an important activator of T cells and NK cells, and (ii) promotion, by the secretory REIC/Dkk-3 protein, of dendritic cell polarization from monocytes. These unique features allow Ad-REIC to exert effective and selective cancer-preventative effects in the manner of an anticancer vaccine. However, the question of how the REIC/Dkk-3 protein leverages anticancer immunity has remained to be answered. We herein report a novel function of the extracellular REIC/Dkk-3―namely, regulation of an immune checkpoint via modulation of PD-L1 on the cancer-cell surface. First, we identified novel interactions of REIC/Dkk-3 with the membrane proteins C5aR, CXCR2, CXCR6, and CMTM6. These proteins all functioned to stabilize PD-L1 on the cell surface. Due to the dominant expression of CMTM6 among the proteins in cancer cells, we next focused on CMTM6 and observed that REIC/Dkk-3 competed with CMTM6 for PD-L1, thereby liberating PD-L1 from its complexation with CMTM6. The released PD-L1 immediately underwent endocytosis-mediated degradation. These results will enhance our understanding of not only the physiological nature of the extracellular REIC/Dkk-3 protein but also the Ad-REIC-mediated anticancer effects. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 0021-924X 174 6 2023 Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration 533 548 EN Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences May Tha Zin Phoo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ikuko Miyazaki Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Translational Research and Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masato Asanuma Department of Medical Neurobiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson’s disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 14 2024 Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface 1371342 EN Tetta Takahashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Faculty of Medicine, Udayana University Youyi Chen Department of Breast Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiki Ochi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshihiko Sakaguchi Department of Microbiology, Tokushima Bunri University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Tumor Pathology, Near InfraRed Photo-Immuno-Therapy Research Institute, Kansai Medical University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshito Zamami Department of Pharmacy, Okayama University Hospital Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein–protein interactions by an immunoprecipitation–Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion. No potential conflict of interest relevant to this article was reported.

Frontiers Media Acta Medica Okayama 2234-943X 13 2023 LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells 1142886 EN Daisuke Hirabayashi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Maruyama Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jin Zhou Medical Oncology Department of Gastrointestinal Tumors, Liaoning Cancer Hospital & Institute, Cancer Hospital of the Dalian University of Technology I. Made Winarsa Ruma Faculty of Medicine, Udayana University I. Wayan Sumardika Faculty of Medicine, Udayana University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Background: EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. Methods: We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (Delta Zn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. Results: MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (Delta Zn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. Conclusions: These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2306-5354 9 11 2022 Functional Blockage of S100A8/A9 Ameliorates Ischemia-Reperfusion Injury in the Lung 673 EN Kentaro Nakata Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mikio Okazaki Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tomohisa Sakaue Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuhei Komoda Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine Dai Shimizu Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Haruchika Yamamoto Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network Shin Tanaka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kentaroh Miyoshi Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshiaki Ohara Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichiro Sugimoto Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masaomi Yamane Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Matsukawa Department of Pathology and Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (1) Background: Lung ischemia-reperfusion (IR) injury increases the mortality and morbidity of patients undergoing lung transplantation. The objective of this study was to identify the key initiator of lung IR injury and to evaluate pharmacological therapeutic approaches using a functional inhibitor against the identified molecule. (2) Methods: Using a mouse hilar clamp model, the combination of RNA sequencing and histological investigations revealed that neutrophil-derived S100A8/A9 plays a central role in inflammatory reactions during lung IR injury. Mice were assigned to sham and IR groups with or without the injection of anti-S100A8/A9 neutralizing monoclonal antibody (mAb). (3) Results: Anti-S100A8/A9 mAb treatment significantly attenuated plasma S100A8/A9 levels compared with control IgG. As evaluated by oxygenation capacity and neutrophil infiltration, the antibody treatment dramatically ameliorated the IR injury. The gene expression levels of cytokines and chemokines induced by IR injury were significantly reduced by the neutralizing antibody. Furthermore, the antibody treatment significantly reduced TUNEL-positive cells, indicating the presence of apoptotic cells. (4) Conclusions: We identified S100A8/A9 as a novel therapeutic target against lung IR injury. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 23 18 2022 Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells 10300 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hidenori Wake Department of Pharmacology, Kindai University Faculty of Medicine Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University I. Made Winarsa Ruma Faculty of Medicine, Udayana University Ken Suzawa Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuma Gohara Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-Ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Faculty of Medicine, Udayana University Youyi Chen Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Futoshi Kuribayashi Department of Biochemistry, Kawasaki Medical School Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Department of General Thoracic Surgery and Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masahiro Nishibori Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis. No potential conflict of interest relevant to this article was reported.

FRONTIERS MEDIA SA Acta Medica Okayama 2234-943X 12 2022 Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading 869393 EN Ai Miyamoto Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Tomoko Honjo Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mirei Masui Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-targeted Therapy, Okayama University Kazuhiro Kakimi Department of Immunotherapeutics, The University of Tokyo Hospital Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Serum autoantibody to cancer/testis antigens (CTAs) is a critical biomarker that reflects the antitumor immune response. Quantitative and multiplexed anti-CTA detection arrays can assess the immune status in tumors and monitor therapy-induced antitumor immune reactions. Most full-length recombinant CTA proteins tend to aggregate. Cysteine residue-specific S-cationization techniques facilitate the preparation of water-soluble and full-length CTAs. Combined with Luminex technology, we designed a multiple S-cationized antigen-immobilized bead array (MUSCAT) assay system to evaluate multiple serum antibodies to CTAs. Reducible S-alkyl-disulfide-cationized antigens in cytosolic conditions were employed to develop rabbit polyclonal antibodies as positive controls. These control antibodies sensitively detected immobilized antigens on beads and endogenous antigens in human lung cancer-derived cell lines. Rabbit polyclonal antibodies successfully confirmed the dynamic ranges and quantitative MUSCAT assay results. An immune monitoring study was conducted using the serum samples on an adenovirus-mediated REIC/Dkk-3 gene therapy clinical trial that showed a successful clinical response in metastatic castration-resistant prostate cancer. Autoantibody responses were closely related to clinical outcomes. Notably, upregulation of anti-CTA responses was monitored before tumor regression. Thus, quantitative monitoring of anti-CTA antibody biomarkers can be used to evaluate the cancer-immunity cycle. A quality-certified serum autoantibody monitoring system is a powerful tool for developing and evaluating cancer immunotherapy. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4425 13 2 2022 Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice 285 EN Ruizhi Xue Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Wenfeng Lin Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jingkai Sun Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Ochiai Laboratory of Veterinary Hygiene, Nippon Veterinary and Life Science University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Zhengyan Tang Department of Urology, Xiangya Hospital, Central South University Peng Huang Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasutomo Nasu Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 +/- 8.12% vs. 23.26 +/- 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 +/- 0.69 vs. 8.27 +/- 0.87, x10(6)), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 +/- 1.55% vs. 72.50 +/- 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 22 2020 Neuroplastinβ-mediated upregulation of solute carrier family 22 member 18 antisense (SLC22A18AS) plays a crucial role in the epithelial-mesenchymal transition, leading to lung cancer cells' enhanced motility 100768 EN Karolina Bajkowska Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ni Luh Gede Yoni Komalasari Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Fan Jiang Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School I. Made Winarsa Ruma University of Surrey Carlos Ichiro Kasano-Camones Faculty of Science and Technology, Division of Molecular Science, Gunma University Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Our recent study revealed an important role of the neuroplastin (NPTN)β downstream signal in lung cancer dissemination in the lung. The molecular mechanism of the signal pathway downstream of NPTNβ is a serial activation of the key molecules we identified: tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) adaptor, nuclear factor (NF)IA/NFIB heterodimer transcription factor, and SAM pointed-domain containing ETS transcription factor (SPDEF). The question of how dissemination is controlled by SPDEF under the activated NPTNβ has not been answered. Here, we show that the NPTNβ-SPDEF-mediated induction of solute carrier family 22 member 18 antisense (SLC22A18AS) is definitely required for the epithelial-mesenchymal transition (EMT) through the NPTNβ pathway in lung cancer cells. In vitro, the induced EMT is linked to the acquisition of active cellular motility but not growth, and this is correlated with highly disseminative tumor progression in vivo. The publicly available data also show the poor survival of SLC22A18AS-overexpressing lung cancer patients. Taken together, these data highlight a crucial role of SLC22A18AS in lung cancer dissemination, which provides novel input of this molecule to the signal cascade of NPTNβ. Our findings contribute to a better understanding of NPTNβ-mediated lung cancer metastasis. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 19365233 13 4 2020 S100 Soil Sensor Receptors and Molecular Targeting Therapy Against Them in Cancer Metastasis 100753 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences The molecular mechanisms underlying the 'seed and soil' theory are unknown. S100A8/A9 (a heterodimer complex of S100A8 and S100A9 proteins that exhibits a 'soil signal') is a ligand for Toll-like receptor 4, causing distant melanoma cells to approach the lung as a 'seeding' site. Unknown soil sensors for S100A8/A9 may exist, e.g., extracellular matrix metalloproteinase inducer, neuroplastin, activated leukocyte cell adhesion molecule, and melanoma cell adhesion molecule. We call these receptor proteins 'novel S100 soil sensor receptors (novel SSSRs).' Here we review and summarize a crucial role of the S100A8/A9-novel SSSRs' axis in cancer metastasis. The binding of S100A8/A9 to individual SSSRs is important in cancer metastasis via upregulations of the epithelial-mesenchymal transition, cellular motility, and cancer cell invasiveness, plus the formation of an inflammatory immune suppressive environment in metastatic organ(s). These metastatic cellular events are caused by the SSSR-featured signal transductions we identified that provide cancer cells a driving force for metastasis. To deprive cancer cells of these metastatic forces, we developed novel biologics that prevent the interaction of S100A8/A9 with SSSRs, followed by the efficient suppression of S100A8/A9-mediated lung-tropic metastasis in vivo. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 24055808 18 2019 Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue 100619 EN Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I. Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 1522-8002 21 7 2019 Critical role of the MCAM-ETV4 axis triggered by extracellular S100A8/A9 in breast cancer aggressiveness 627 640 EN Youyi Chen Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences I Wayan Sumardika Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nahoko Tomonobu Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yusuke Inoue Faculty of Science and Technology, Division of Molecular Science, Gunma University Hidekazu Iioka Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Yosuke Mitsui Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken Saito Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences I Made Winarsa Ruma Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroki Sato Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akira Yamauchi Department of Biochemistry, Kawasaki Medical School Hitoshi Murata Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ken-ichi Yamamoto Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Shuta Tomida Department of Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Shien Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromasa Yamamoto Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichi Soh Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miyoko Kubo Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Endy Widya Putranto Department of Pediatrics, Dr. Sardjito Hospital/Faculty of Medicine, Universitas Gadjah Mada Takashi Murakami Department of Microbiology, Faculty of Medicine, Saitama Medical University Ming Liu Department of General Surgery & Bio-Bank of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University Toshihiko Hibino Department of Dermatology, Tokyo Medical University Masahiro Nishibori Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Eisaku Kondo Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences Shinichi Toyooka Departments of Thoracic, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes. No potential conflict of interest relevant to this article was reported.