Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche

Koike N., Kota R., Naito Y., Hayakawa N., Matsuura T., Hishiki T., Onishi N., Fukada J., Suematsu M., Shigematsu N., Saya H., Sampetrean O.

Communications Biology (Communications Biology) 3 ( 1 ) 2020.12

© 2020, The Author(s). Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.

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Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell

Tanno N., Kuninaka S., Fujimura S., Takemoto K., Okamura K., Takeda N., Araki K., Araki M., Saya H., Ishiguro K.i.

Scientific Reports (Scientific Reports) 10 ( 1 ) 2020.12

© 2020, The Author(s). FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

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Downregulation of the CCL2/CCR2 and CXCL10/CXCR3 axes contributes to antitumor effects in a mouse model of malignant glioma

Shono K., Yamaguchi I., Mizobuchi Y., Kagusa H., Sumi A., Fujihara T., Nakajima K., Kitazato K.T., Matsuzaki K., Saya H., Takagi Y.

Scientific Reports (Scientific Reports) 10 ( 1 ) 2020.12

© 2020, The Author(s). Glioblastoma multiforme involves glioma stem cells (GSCs) that are resistant to various therapeutic approaches. Here, we studied the importance of paracrine signaling in the glioma microenvironment by focusing on the celecoxib-mediated role of chemokines C–C motif ligand 2 (CCL2), C-X-C ligand 10 (CXCL10), and their receptors, CCR2 and CXCR3, in GSCs and a GSC-bearing malignant glioma model. C57BL/6 mice were injected with orthotopic GSCs intracranially and divided into groups administered either 10 or 30 mg/kg celecoxib, or saline to examine the antitumor effects associated with chemokine expression. In GSCs, we analyzed cell viability and expression of chemokines and their receptors in the presence/absence of celecoxib. In the malignant glioma model, celecoxib exhibited antitumor effects in a dose dependent manner and decreased protein and mRNA levels of Ccl2 and CxcL10 and Cxcr3 but not of Ccr2. CCL2 and CXCL10 co-localized with Nestin+ stem cells, CD16+ or CD163+ macrophages and Iba-1+ microglia. In GSCs, celecoxib inhibited Ccl2 and Cxcr3 expression in a nuclear factor-kappa B-dependent manner but not Ccr2 and CxcL10. Moreover, Ccl2 silencing resulted in decreased GSC viability. These results suggest that celecoxib-mediated regulation of the CCL2/CCR2 and CXCL10/ CXCR3 axes may partially contribute to glioma-specific antitumor effects.

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Molecular and cellular mechanisms underlying brain metastasis of breast cancer

Hosonaga M., Saya H., Arima Y.

Cancer and Metastasis Reviews (Cancer and Metastasis Reviews) 39 ( 3 ) 711 - 720 2020.09

ISSN 01677659

© 2020, The Author(s). Metastasis of cancer cells to the brain occurs frequently in patients with certain subtypes of breast cancer. In particular, patients with HER2-positive or triple-negative breast cancer are at high risk for the development of brain metastases. Despite recent advances in the treatment of primary breast tumors, the prognosis of breast cancer patients with brain metastases remains poor. A better understanding of the molecular and cellular mechanisms underlying brain metastasis might be expected to lead to improvements in the overall survival rate for these patients. Recent studies have revealed complex interactions between metastatic cancer cells and their microenvironment in the brain. Such interactions result in the activation of various signaling pathways related to metastasis in both cancer cells and cells of the microenvironment including astrocytes and microglia. In this review, we focus on such interactions and on their role both in the metastatic process and as potential targets for therapeutic intervention.

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Targeting of cancer stem cells by differentiation therapy

Arima Y., Nobusue H., Saya H.

Cancer Science (Cancer Science) 111 ( 8 ) 2689 - 2695 2020.08

ISSN 13479032

© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Chemoresistance is a hallmark of cancer stem cells (CSCs). To develop novel therapeutic strategies that target CSCs, we established osteosarcoma-initiating (OSi) cells by introducing the c-Myc gene into bone marrow stromal cells derived from Ink4a/Arf KO mice. These OSi cells include bipotent committed cells (similar to osteochondral progenitor cells) with a high tumorigenic activity as well as tripotent cells (similar to mesenchymal stem cells) of low tumorigenicity. We recently showed that the tripotent OSi cells are highly resistant to chemotherapeutic agents, and that depolymerization of the actin cytoskeleton in these cells induces their terminal adipocyte differentiation and suppresses their tumorigenicity. We here provide an overview of modulation of actin cytoskeleton dynamics associated with terminal adipocyte differentiation in osteosarcoma as well as discuss the prospects for new therapeutic strategies that target chemoresistant CSCs by inducing their differentiation.

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The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics

Kunitomi H., Oki Y., Onishi N., Kano K., Banno K., Aoki D., Saya H., Nobusue H.

Genes to Cells (Genes to Cells) 25 ( 3 ) 165 - 174 2020.03

ISSN 13569597

© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd Adipocyte differentiation is accompanied by a pronounced change in the actin cytoskeleton characterized by the reorganization of filamentous (F)-actin stress fibers into cortical F-actin structures. We previously showed that depolymerization of F-actin stress fibers induced by inactivation of RhoA–ROCK (Rho-associated kinase) signaling acts as a trigger for adipocyte differentiation. The relevance and underlying mechanism of the formation of cortical F-actin structures from depolymerized actin during adipocyte differentiation have remained unclear, however. We have now examined the mechanistic relation between actin dynamics and adipogenic induction. Transient exposure to the actin-depolymerizing agent latrunculin A (LatA) supported the formation of adipocyte-associated cortical actin structures and the completion of terminal adipocyte differentiation in the presence of insulin, whereas long-term exposure to LatA prevented such actin reorganization as well as terminal adipogenesis. Moreover, these effects of insulin were prevented by inhibition of phosphatidylinositol 3-kinase (PI3K)–Rac1 signaling and the actin-related protein 2/3 (Arp2/3) complex which is a critical component of the cortical actin networks. Our findings thus suggest that the insulin-PI3K-Rac1 axis leads to the formation of adipocyte-associated cortical actin structures which is essential for the completion of adipocyte differentiation.

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Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect

Tamura R., Miyoshi H., Morimoto Y., Oishi Y., Sampetrean O., Iwasawa C., Mine Y., Saya H., Yoshida K., Okano H., Toda M.

Human Gene Therapy (Human Gene Therapy) 31 ( 5-6 ) 352 - 366 2020.03

ISSN 10430342

© Copyright 2020, by Mary Ann Liebert, Inc., publishers 2020. Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.

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Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma

Padthaisong S., Thanee M., Namwat N., Phetcharaburanin J., Klanrit P., Khuntikeo N., Titapun A., Sungkhamanon S., Saya H., Loilome W.

Journal of Translational Medicine (Journal of Translational Medicine) 18 ( 1 ) 2020.02

© 2020 The Author(s). Background: Cancer recurrence is the important problem of cholangiocarcinoma (CCA) patients, lead to a very high mortality rate. Therefore, the identification of candidate markers to predict CCA recurrence is needed in order to effectively manage the disease. This study aims to examine the predictive value of cancer stem cell (CSC) markers on the progression and recurrence of CCA patients. Methods: The expression of 6 putative CSC markers, cluster of differentiation 44 (CD44), CD44 variant 6 (CD44v6), CD44 variants 8-10 (CD44v8-10), cluster of differentiation 133 (CD133), epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase 1A1 (ALDH1A1), was investigated in 178 CCA tissue samples using immunohistochemistry (IHC) and analyzed with respect to clinicopathological data and patient outcome including recurrence-free survival (RFS) and overall survival (OS). The candidate CSC markers were also investigated in serum from CCA patients, and explored for their predictive ability on CCA recurrence. Results: Elevated protein level of CD44 and positive expression of CD44v6 and CD44v8-10 were significantly associated with short RFS and OS, while high levels of ALDH1A1 were correlated with a favorable prognosis patient. The elevated CD44v6 level was also correlated with higher tumor staging, whereas a decreasing level of ALDH1A1 was correlated with lower tumor staging. The levels of CD44, CD44v6 and CD44v8-10 were also correlated and were associated with a poor outcome. Furthermore, soluble CD44, CD44v6, CD44v8-10 and EpCAM were significantly increased in the recurrence group for early stage CCA; they also correlated with high levels of the tumor marker CA19-9. Elevated levels of CD44, CD44v6, CD44v8-10 or EpCAM alone or in combination has the potential to predict CCA recurrence. Conclusions: The overexpression of CD44, CD44v6, CD44v8-10 and EpCAM increases predictability of post-operative CCA recurrence. Moreover, the overexpression of the panel of CSC markers combined with CA19-9 could improve our predictive ability for tumor recurrence in early stage CCA patients. This result may be beneficial for the patients in order to predict the outcome after treatment and may be useful for clinical intervention in order to improve patient survival.

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A potent blood–brain barrier-permeable mutant IDH1 inhibitor suppresses the growth of glioblastoma with IDH1 mutation in a patient-derived orthotopic xenograft model

Machida Y., Nakagawa M., Matsunaga H., Yamaguchi M., Ogawara Y., Shima Y., Yamagata K., Katsumoto T., Hattori A., Itoh M., Seki T., Nishiya Y., Nakamura K., Suzuki K., Imaoka T., Baba D., Suzuki M., Sampetrean O., Saya H., Ichimura K., Kitabayashi I.

Molecular Cancer Therapeutics (Molecular Cancer Therapeutics) 19 ( 2 ) 375 - 383 2020.02

ISSN 15357163

©2019 American Association for Cancer Research. Gliomas are the second most common primary brain tumors in adults. They are treated with combination therapies, including surgery, radiotherapy, and chemotherapy. There are currently limited treatment options for recurrent gliomas, and new targeted therapies need to be identified, especially in glioblastomas, which have poor prognosis. Isocitrate dehydrogenase (IDH) mutations are detected in various tumors, including gliomas. Most patients with IDH mutant glioma harbor the IDH1R132H subtype. Mutant IDH catalyzes the conversion of a-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which induces aberrant epigenetic status and contributes to malignant progression, and is therefore a potential therapeutic target for IDH mutant tumors. The present study describes a novel, orally bioavailable selective mutant IDH1 inhibitor, DS-1001b. The drug has high blood–brain barrier (BBB) permeability and inhibits IDH1R132H. Continuous administration of DS-1001b impaired tumor growth and decreased 2-HG levels in subcutaneous and intracranial xenograft models derived from a patient with glioblastoma with IDH1 mutation. Moreover, the expression of glial fibrillary acidic protein was strongly induced by DS-1001b, suggesting that inhibition of mutant IDH1 promotes glial differentiation. These results reveal the efficacy of BBB-permeable DS-1001b in orthotopic patient-derived xenograft models and provide a preclinical rationale for the clinical testing of DS-1001b in recurrent gliomas.

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Identification of kit-ligand a as the gene responsible for the medaka pigment cell mutant few melanophore

Otsuki Y., Okuda Y., Naruse K., Saya H.

G3: Genes, Genomes, Genetics (G3: Genes, Genomes, Genetics) 10 ( 1 ) 311 - 319 2020.01

Copyright © 2020 Otsuki et al. The body coloration of animals is due to pigment cells derived from neural crest cells, which are multipotent and differentiate into diverse cell types. Medaka (Oryzias latipes) possesses four distinct types of pigment cells known as melanophores, xanthophores, iridophores, and leucophores. The few melanophore (fm) mutant of medaka is characterized by reduced numbers of melanophores and leucophores. We here identify kit-ligand a (kitlga) as the gene whose mutation gives rise to the fm phenotype. This identification was confirmed by generation of kitlga knockout medaka and the findings that these fish also manifest reduced numbers of melanophores and leucophores and fail to rescue the fm mutant phenotype. We also found that expression of sox5, pax7a, pax3a, and mitfa genes is down-regulated in both fm and kitlga knockout medaka, implicating c-Kit signaling in regulation of the expression of these genes as well as the encoded transcription factors in pigment cell specification. Our results may provide insight into the pathogenesis of c-Kit–related pigmentation disorders such as piebaldism in humans, and our kitlga knockout medaka may prove useful as a tool for drug screening.

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The DNA topoisomerase II inhibitor amsacrine as a novel candidate adjuvant in a model of glaucoma filtration surgery

Yamamoto K., Kokubun T., Sato K., Akaishi T., Shimazaki A., Nakamura M., Shiga Y., Tsuda S., Omodaka K., Saya H., Nakazawa T.

Scientific Reports (Scientific Reports) 9 ( 1 ) 2019.12

© 2019, The Author(s). Treatments for refractory glaucoma include trabeculectomy, in which a filtering bleb is created to reduce aqueous pressure. Mitomycin C (MMC) is often used as an adjuvant to reduce post-trabeculectomy bleb scarring and consequent failure. However, scarring sometimes still occurs. Thus, we searched for more effective trabeculectomy adjuvants with high-throughput screening (HTS) of a library of 1,165 off-patent drug compounds. This revealed that amsacrine (AMSA), a DNA topoisomerase II (TOP2) inhibitor, was the top candidate. Compared to MMC, rabbits that underwent trabeculectomy with 10% AMSA had lower IOP at 42, 56, and 70 days (P < 0.01 at all measurement points) and a higher bleb score at 28, 42, 56, and 70 days (P = < 0.01, 0.04, 0.04, and < 0.01, respectively). Compared to saline, rabbits that received 1% AMSA also had lower IOP and better bleb score at all time points, without a sharp drop in IOP just after surgery (all P < 0.01). Both effects were milder than MMC at 7 days (P = 0.02 and <0.01, respectively). Thus, this study showed that HTS may help identify new, promising uses for off-patent drugs. Furthermore, trabeculectomy with AMSA at a suitable concentration may improve the prognosis after trabeculectomy compared to MMC.

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Ropinirole hydrochloride remedy for amyotrophic lateral sclerosis – Protocol for a randomized, double-blind, placebo-controlled, single-center, and open-label continuation phase I/IIa clinical trial (ROPALS trial)

Morimoto S., Takahashi S., Fukushima K., Saya H., Suzuki N., Aoki M., Okano H., Nakahara J.

Regenerative Therapy (Regenerative Therapy) 11 143 - 166 2019.12

© 2019 The Japanese Society for Regenerative Medicine Introduction: Amyotrophic lateral sclerosis (ALS) is an intractable and incurable neurological disease. It is a progressive disease characterized by muscle atrophy and weakness caused by selective vulnerability of upper and lower motor neurons. In disease research, it has been common to use mouse models carrying mutations in responsible genes for familial ALS as pathological models of ALS. However, there is no model that has reproduced the actual conditions of human spinal cord pathology. Thus, we developed a method of producing human spinal motor neurons using human induced pluripotent stem cells (iPSCs) and an innovative experimental technique for drug screening. As a result, ropinirole hydrochloride was eventually discovered after considering such results as its preferable transitivity in the brain and tolerability, including possible adverse reactions. Therefore, we explore the safety, tolerability and efficacy of ropinirole hydrochloride as an ALS treatment in this clinical trial. Methods: The ROPALS trial is a single-center double-blind randomized parallel group-controlled trial of the safety, tolerability, and efficacy of the ropinirole hydrochloride extended-release tablet (Requip CR) at 2- to 16-mg doses in patients with ALS. Twenty patients will be recruited for the active drug group (fifteen patients) and placebo group (five patients). All patients will be able to receive the standard ALS treatment of riluzole if not changed the dosage during this trial. The primary outcome will be safety and tolerability at 24 weeks, defined from the date of randomization. Secondary outcome will be the efficacy, including any change in the ALS Functional Rating Scale-Revised (ALSFRS-R), change in the Combined Assessment of Function and Survival (CAFS), and the composite endpoint as a sum of Z-transformed scores on various clinical items. Notably, we will perform an explorative search for a drug effect evaluation using the patient-derived iPSCs to prove this trial concept. Eligible patients will have El Escorial Possible, clinically possible and laboratory-supported, clinically probable, or clinically definite amyotrophic lateral sclerosis with disease duration less than 60 months (inclusive), an ALSFRS-R score ≥2 points on all items and age from 20 to 80 years. Conclusion: Patient recruitment began in December 2018 and the last patient is expected to complete the trial protocol in November 2020. Trial registration: Current controlled trials UMIN000034954 and JMA-IIA00397 Protocol version: version 1.6 (Date; 5/Apr/2019).

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Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma

Okazaki S., Umene K., Yamasaki J., Suina K., Otsuki Y., Yoshikawa M., Minami Y., Masuko T., Kawaguchi S., Nakayama H., Banno K., Aoki D., Saya H., Nagano O.

Cancer Science (Cancer Science) 110 ( 11 ) 3453 - 3463 2019.11

ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.

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Neuroprotective effects of memantine via enhancement of autophagy

Hirano K., Fujimaki M., Sasazawa Y., Yamaguchi A., Ishikawa K., Miyamoto K., Souma S., Furuya N., Imamichi Y., Yamada D., Saya H., Akamatsu W., Saiki S., Hattori N.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 518 ( 1 ) 161 - 170 2019.10

ISSN 0006291X

© 2019 Elsevier Inc. Introduction: Chemical intervention of autophagy has been investigated in clinical trials for various age-related conditions such as sarcopenia and neurodegeneration. However, at present, no autophagy inducer has been established as a disease-modifying agent against neurodegenerative diseases. Methods: We screened a library consisting of 796 medicines clinically approved (in Japan) for autophagy enhancers as potential neurodegeneration therapeutics using HeLa cells stably expressing green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) followed by an analysis of the molecular mechanisms using various neuronal models. Results: The primary screening identified 152 hits in a static cellular state. A widely available Alzheimer's disease drug, memantine, which antagonizes N-Methyl-D-aspartate receptor (NMDAR), was one of the hits. Memantine increased the levels of LC3-II in a dose-dependent and time-dependent manner, and upregulated autophagic flux. In addition, the pharmacological effects of memantine on autophagy were independent of mTORC1 activity and NMDAR activation. Furthermore, a VPS34 inhibitor suppressed the memantine-induced LC3-II upregulation, suggesting that memantine may affect VPS34 complex activity. Notably, intracellular Huntington's disease-specific aggregates of elongated huntingtin, a well-established autophagy substrate, were significantly decreased by memantine. In addition, memantine enhanced elimination of degraded mitochondrial in neurons derived from induced pluripotent stem cells of PARK2 or PARK6 patients, who exhibited defective PINK1/parkin-mediated mitophagy, suggests that memantine accelerated the clearance of damaged mitochondria. Conclusion: These findings indicate that memantine may be beneficial for the treatment of neurodegeneration characterized by the abnormal accumulation of autophagy or mitophagy substrates.

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IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma

Kofuji S., Hirayama A., Eberhardt A., Kawaguchi R., Sugiura Y., Sampetrean O., Ikeda Y., Warren M., Sakamoto N., Kitahara S., Yoshino H., Yamashita D., Sumita K., Wolfe K., Lange L., Ikeda S., Shimada H., Minami N., Malhotra A., Morioka S., Ban Y., Asano M., Flanary V., Ramkissoon A., Chow L., Kiyokawa J., Mashimo T., Lucey G., Mareninov S., Ozawa T., Onishi N., Okumura K., Terakawa J., Daikoku T., Wise-Draper T., Majd N., Kofuji K., Sasaki M., Mori M., Kanemura Y., Smith E., Anastasiou D., Wakimoto H., Holland E., Yong W., Horbinski C., Nakano I., DeBerardinis R., Bachoo R., Mischel P., Yasui W., Suematsu M., Saya H., Soga T., Grummt I., Bierhoff H., Sasaki A.

Nature cell biology (Nature cell biology) 21 ( 8 ) 1003 - 1014 2019.08

ISSN 14657392

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurance of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

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ROCK inhibition induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant osteosarcoma cells

Takahashi N., Nobusue H., Shimizu T., Sugihara E., Yamaguchi-Iwai S., Onishi N., Kunitomi H., Kuroda T., Saya H.

Cancer Research (Cancer Research) 79 ( 12 ) 3088 - 3099 2019.06

ISSN 00085472

© 2019 American Association for Cancer Research. Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stemcells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapyresistant osteosarcoma stem cells.

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Glucose transporter 1-mediated vascular translocation of nanomedicines enhances accumulation and efficacy in solid tumors

Suzuki K., Miura Y., Mochida Y., Miyazaki T., Toh K., Anraku Y., Melo V., Liu X., Ishii T., Nagano O., Saya H., Cabral H., Kataoka K.

Journal of Controlled Release (Journal of Controlled Release) 301 28 - 41 2019.05

ISSN 01683659

© 2019 Elsevier B.V. Nanomedicine modification with ligands directed to receptors on tumor blood vessels has the potential for selectively enhancing nanomedicine accumulation in malignant tissues by overcoming the vascular barrier of tumors. Nevertheless, the development of broadly applicable ligand approaches capable of promoting the transvascular transport of nanomedicines in a wide spectrum of tumors has been elusive so far. By considering the indispensable and persistent glycolytic fueling of tumors, we developed glucose-installed polymeric micelles loading cisplatin (Gluc-CDDP/m) targeting the glucose transporter 1 (GLUT1), which is overexpressed in most tumors and present on vascular endothelial cells, toward improving the delivery efficiency and therapeutic efficacy. The design of the glucose ligands on Gluc-CDDP/m was engineered to control the conjugation via the carbon 6 of the glucose moieties, as well as the ligand density on the poly (ethylene glycol) (PEG) shell of the micelles. The series of micelles was then studied in vitro and in vivo against GLUT1-high human squamous cell carcinoma of the head and neck OSC-19 cells and GLUT1-low human glioblastoma-astrocytoma U87MG cells. Our results showed that precisely tuning the micelles to have glucose ligands on 25% of their PEG chains increased the efficacy against the tumors by significantly enhancing the tumor accumulation, even in GLUT1-low U87MG tumors. The enhancement of the intratumoral levels of these micelles was hindered by concomitant administration of glucose, or the GLUT1 inhibitor STF-31, confirming a GLUT1/glucose-mediated increment of the accumulation. Intravital confocal laser scanning microscopy imaging of tumor tissues further demonstrated the rapid extravasation and penetration of Gluc-CDDP/m in OSC-19 tumors compared to non-targeted CDDP/m. These findings indicate GLUT1-targeting as a promising approach for overcoming the vascular barrier and boosting the delivery of nanomedicine in tumors.

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Visualization of spatiotemporal dynamics of human glioma stem cell invasion

Tamura R., Miyoshi H., Sampetrean O., Shinozaki M., Morimoto Y., Iwasawa C., Fukaya R., Mine Y., Masuda H., Maruyama T., Narita M., Saya H., Yoshida K., Okano H., Toda M.

Molecular brain (Molecular brain) 12 ( 1 ) 2019.05

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

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An evolving story of the metastatic voyage of ovarian cancer cells: cellular and molecular orchestration of the adipose-rich metastatic microenvironment

Motohara T., Masuda K., Morotti M., Zheng Y., El-Sahhar S., Chong K., Wietek N., Alsaadi A., Karaminejadranjbar M., Hu Z., Artibani M., Gonzalez L., Katabuchi H., Saya H., Ahmed A.

Oncogene (Oncogene) 38 ( 16 ) 2885 - 2898 2019.04

ISSN 09509232

© 2018, The Author(s). Metastasis is a complex multistep process that involves critical interactions between cancer cells and a variety of stromal components in the tumor microenvironment, which profoundly influence the different aspects of the metastatic cascade and organ tropism of disseminating cancer cells. Ovarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Evidence has demonstrated that ovarian cancer possesses specific metastatic tropism for the adipose-rich omentum, which has a pivotal role in the creation of the metastatic tumor microenvironment in the intraperitoneal cavity. Considering the distinct biology of ovarian cancer metastasis, the elucidation of the cellular and molecular mechanisms underlying the reciprocal interplay between ovarian cancer cells and surrounding stromal cell types in the adipose-rich metastatic microenvironment will provide further insights into the development of novel therapeutic approaches for patients with advanced ovarian cancer. Herein, we review the biological mechanisms that regulate the highly orchestrated crosstalk between ovarian cancer cells and various cancer-associated stromal cells in the metastatic tumor microenvironment with regard to the omentum by illustrating how different stromal cells concertedly contribute to the development of ovarian cancer metastasis and metastatic tropism for the omentum.

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Sulfasalazine could modulate the CD44v9-xCT system and enhance cisplatin-induced cytotoxic effects in metastatic bladder cancer

Ogihara K., Kikuchi E., Okazaki S., Hagiwara M., Takeda T., Matsumoto K., Kosaka T., Mikami S., Saya H., Oya M.

Cancer Science (Cancer Science) 110 ( 4 ) 1431 - 1441 2019.04

ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The prognostic role of CD44v9, a variant isoform of CD44 and a new cell surface marker of cancer stem cells, remains unclear in bladder cancer (BC) patients. Furthermore, limited information is available on the functional role of sulfasalazine (SSZ), which could modulate the CD44v9-xCT system in order to enhance cisplatin (CDDP)-induced cytotoxicity and inhibit the metastatic potential of BC. CD44v9 protein expression was examined immunohistochemically in 63 muscle invasive BC (MIBC) patients who underwent radical cystectomy. CD44v9 expression was independently associated with disease recurrence and cancer-specific death in MIBC. Cytotoxic effects, glutathione levels, and reactive oxygen species production by SSZ and CD44v9 and phospho-p38 MAPK protein expression by SSZ with or without CDDP were assessed in MBT-2V cells with highly metastatic potential. Sulfasalazine exerted cytotoxic effects against MBT-2V cells by inhibiting glutathione levels and inducing the production of reactive oxygen species. Sulfasalazine in combination with CDDP appeared to exert strong cytotoxic effects against MBT-2V cells by inhibiting CD44v9 expression and upregulating phospho-p38 MAPK expression. The inhibitory effects of SSZ with or without CDDP were also investigated using an MBT-2V lung metastatic model. In the murine lung metastatic BC model, SSZ significantly prolonged animal survival. Furthermore, the combination of SSZ with CDDP exerted stronger inhibitory effects on the establishment of lung tumor nodules than SSZ or CDDP alone. CD44v9 expression could be a clinical biomarker for predicting poor outcomes in MIBC patients. Sulfasalazine in combination with CDDP has potential as a novel therapy against metastatic BC.

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