Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Modeling phenotypes of malignant gliomas

Sampetrean Oltea, Saya Hideyuki

Cancer Science (Cancer Science) 109 ( 1 ) 6 - 14 2018.01

ISSN 1347-9032

Malignant gliomas are primary tumors of the central nervous system characterized by diffuse infiltration into the brain and a high recurrence rate. Advances in comprehensive genomic studies have provided unprecedented insight into the genetic and molecular heterogeneity of these tumors and refined our understanding of their evolution from low to high grade. However, similar levels of phenotypic characterization are indispensable to understanding the complexity of malignant gliomas. Experimental glioma models have also achieved great progress in recent years. Advances in transgenic technologies and cell culture have allowed the establishment of mouse models that mirror the human disease with increasing fidelity and which support single-cell resolution for phenotypic analyses. Here we review the major types of preclinical glioma models, with an emphasis on how recent developments in experimental modeling have shed new light on two fundamental aspects of glioma phenotype, their cell of origin and their invasive potential.

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Stat3 as a potential therapeutic target for rheumatoid arthritis

Oike Takatsugu, Sato Yuiko, Kobayashi Tami, Miyamoto Kana, Nakamura Satoshi, Kaneko Yosuke, Kobayashi Shu, Harato Kengo, Saya Hideyuki, Matsumoto Morio, Nakamura Masaya, Niki Yasuo, Miyamoto Takeshi

Scientific Reports 7 ( 1 ) 2017.12

ISSN 2045-2322

Rheumatoid arthritis (RA) is a multi-factorial disease characterized by chronic inflammation and destruction of multiple joints. To date, various biologic treatments for RA such as anti-Tumor necrosis factor alpha antibodies have been developed; however, mechanisms underlying RA development remain unclear and targeted therapy for this condition has not been established. Here, we provide evidence that signal transducer and activator of transcription 3 (Stat3) promotes inflammation and joint erosion in a mouse model of arthritis. Stat3 global KO mice show early embryonic lethality; thus, we generated viable Stat3 conditional knockout adult mice and found that they were significantly resistant to collagen-induced arthritis (CIA), the most common RA model, compared with controls. We then used an in vitro culture system to screen ninety-six existing drugs to select Stat3 inhibitors and selected five candidate inhibitors. Among them, three significantly inhibited development of arthritis and joint erosion in CIA wild-Type mice. These findings suggest that Stat3 inhibitors may serve as promising drugs for RA therapy.

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Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality

Kitagawa Mayumi, Liao Pei Ju, Lee Kyung Hee, Wong Jasmine, Shang See Cheng, Minami Noriaki, Sampetrean Oltea, Saya Hideyuki, Lingyun Dai, Prabhu Nayana, Diam Go Ka, Sobota Radoslaw, Larsson Andreas, Nordlund Pär, McCormick Frank, Ghosh Sujoy, Epstein David M., Dymock Brian W., Lee Sang Hyun

Nature Communications 8 ( 1 ) 2017.12

ISSN 2041-1723

Achieving robust cancer-specific lethality is the ultimate clinical goal. Here, we identify a compound with dual-inhibitory properties, named a131, that selectively kills cancer cells, while protecting normal cells. Through an unbiased CETSA screen, we identify the PIP4K lipid kinases as the target of a131. Ablation of the PIP4Ks generates a phenocopy of the pharmacological effects of PIP4K inhibition by a131. Notably, PIP4Ks inhibition by a131 causes reversible growth arrest in normal cells by transcriptionally upregulating PIK3IP1, a suppressor of the PI3K/Akt/mTOR pathway. Strikingly, Ras activation overrides a131-induced PIK3IP1 upregulation and activates the PI3K/Akt/mTOR pathway. Consequently, Ras-transformed cells override a131-induced growth arrest and enter mitosis where a131's ability to de-cluster supernumerary centrosomes in cancer cells eliminates Ras-activated cells through mitotic catastrophe. Our discovery of drugs with a dual-inhibitory mechanism provides a unique pharmacological strategy against cancer and evidence of cross-activation between the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR pathways via a RasPI3K signaling network.

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Reducing inflammatory cytokine production from renal collecting duct cells by inhibiting GATA2 ameliorates acute kidney injury

Yu Lei, Moriguchi Takashi, Kaneko Hiroshi, Hayashi Makiko, Hasegawa Atsushi, Nezu Masahiro, Saya Hideyuki, Yamamoto Masayuki, Shimizu Ritsuko

Molecular and Cellular Biology 37 ( 22 ) 2017.11

ISSN 0270-7306

Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia reperfusion injury (IRI). Notably, G2CKO mice exhibited less severe kidney damage, with reduced granulomacrophagic infiltration upon IRI. Transcriptome analysis revealed that a series of inflammatory cytokine genes were downregulated in GATA2-deficient CD cells, suggesting that GATA2 induces inflammatory cytokine expression in diseased kidney CD cells. Through high-throughput chemical library screening, we identified a potent GATA inhibitor. The chemical reduces cytokine production in CD cells and protects the mouse kidney from IRI. These results revealed a novel pathological mechanism of renal IRI, namely, that CD cells produce inflammatory cytokines and promote IRI progression. In injured kidney CD cells, GATA2 exerts a proinflammatory function by upregulating inflammatory cytokine gene expression. GATA2 can therefore be considered a therapeutic target for AKI.

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CD44 variant–dependent regulation of redox balance in EGFR mutation–positive non–small cell lung cancer

Kawano Yuko, Iwama Eiji, Tsuchihashi Kenji, Shibahara Daisuke, Harada Taishi, Tanaka Kentaro, Nagano Osamu, Saya Hideyuki, Nakanishi Yoichi, Okamoto Isamu

Lung Cancer 113 72 - 78 2017.11

ISSN 0169-5002

Objectives The regulation of redox balance in cancer cells is an important factor in tumor development and chemoresistance, with oncogene activation having been shown to induce the generation of reactive oxygen species (ROS). Activating mutations of the epidermal growth factor receptor gene (EGFR) are oncogenic drivers in non–small cell lung cancer (NSCLC), but it has remained unknown whether ligand-independent EGFR signaling conferred by EGFR mutation triggers ROS generation in NSCLC cells. Materials and Methods HEK293T cells were transfected with an expression vector for mutant EGFR. The expression of CD44 variant (CD44v) isoforms in NSCLC cell lines was evaluated by flow cytometry. Cells were depleted of CD44v by RNA interference and assayed for ROS and glutathione (GSH) levels. The effect of CD44v on cisplatin sensitivity was evaluated in vitro with the MTS assay. Results EGFR signaling due to EGFR mutation increased ROS levels in transfected HEK293T cells. The expression of CD44v isoforms was found to be inversely correlated with basal ROS levels in EGFR mutation–positive NSCLC cell lines. Knockdown of CD44v induced depletion of intracellular GSH and increased ROS levels in EGFR-mutated NSCLC cells that express CD44v at a high level (CD44v^high). In addition, depletion of GSH by treatment with buthionine-[S, R]-sulfoximine induced marked accumulation of ROS and enhanced the cytotoxicity of cisplatin in CD44v^high EGFR-mutated NSCLC cells but not in corresponding CD44v^low cells. This enhancement of cisplatin cytotoxicity by GSH depletion was prevented by treatment with the antioxidant N-acetyl-L-cysteine. Knockdown of CD44v also enhanced cisplatin cytotoxicity in CD44v^high EGFR mutation–positive NSCLC cells but not in CD44v^low cells. Conclusion Our results thus implicate CD44v in redox adaptation and as a potential target for treatment in CD44v^high EGFR-mutated NSCLC cells.

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Organotypic brain explant culture as a drug evaluation system for malignant brain tumors

Minami Noriaki, Maeda Yusuke, Shibao Shunsuke, Arima Yoshimi, Ohka Fumiharu, Kondo Yutaka, Maruyama Koji, Kusuhara Masatoshi, Sasayama Takashi, Kohmura Eiji, Saya Hideyuki, Sampetrean Oltea

Cancer Medicine 6 ( 11 ) 2635 - 2645 2017.11

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H-Ras^V12 in Ink4a/Arf^−/− neural stem/progenitor cells to establish tumor-bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence-based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel-specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real-time imaging of the microvascular network and complemented time-lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.

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Establishment of pten knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

Matsuzaki Yuriko, Sakuma Tetsushi, Yamamoto Takashi, Saya Hideyuki

PLoS One 12 ( 10 ) 2017.10

ISSN 1932-6203

Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization (hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

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Convection-enhanced delivery of sulfasalazine prolongs survival in a glioma stem cell brain tumor model

Haryu Shinya, Saito Ryuta, Jia Wenting, Shoji Takuhiro, Mano Yui, Sato Aya, Kanamori Masayuki, Sonoda Yukihiko, Sampetrean Oltea, Saya Hideyuki, Tominaga Teiji

Journal of Neuro-Oncology (Journal of Neuro-Oncology) 136 ( 1 ) 1 - 9 2017.09

ISSN 0167-594X

Expression of CD44 in glioma cells was previously correlated with tumor grade and is considered a stem cell marker. CD44 stabilizes the cystine–glutamate transporter (xCT) and inhibits apoptosis in cancer stem cells (CSCs). Recently it was found that Sulfasalazine (SSZ), an anti-inflammatory drug, acts as an inhibitor of xCT and therefore has potential as a targeted therapy for CSCs. In this study, we tested an efficacy of SSZ against glioma stem cell model developed in rats. As poor penetration of blood–brain barrier resulted in insufficient efficacy of systemic SSZ treatment, SSZ was delivered locally with convection-enhanced delivery (CED). In vitro, expression of CD44 in glioma cells and efficacy of SSZ against glioma cells and glioma stem cells were confirmed. SSZ demonstrated anti-proliferative activity in a dose dependent manner against these cells. This activity was partially reversible with the addition of antioxidant, N-acetyl-l-cysteine, to the medium. In vivo, CED successfully delivered SSZ into the rat brain parenchyma. When delivered at 5 mM concentration, which was the highest possible concentration when SSZ was dissolved in water, CED of SSZ resulted in almost no tissue damage. Against highly malignant bRiTs-G3 brain tumor xenografted rat model; the glioma stem cell model, CED of SSZ at 5 mM concentration induced apoptosis and prolonged survival. Consequently, CED of SSZ induced glioma stem cell death without evidence of tissue damage to normal brain parenchyma. This strategy may be a promising targeted treatment against glioma stem cells.

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cRGD peptide installation on cisplatin-loaded nanomedicines enhances efficacy against locally advanced head and neck squamous cell carcinoma bearing cancer stem-like cells

Miyano Kazuki, Cabral Horacio, Miura Yutaka, Matsumoto Yu, Mochida Yuki, Kinoh Hiroaki, Iwata Caname, Nagano Osamu, Saya Hideyuki, Nishiyama Nobuhiro, Kataoka Kazunori, Yamasoba Tatsuya

Journal of Controlled Release 261 275 - 286 2017.09

ISSN 0168-3659

Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses α_vβ₅ integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC.

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Endothelium-induced three-dimensional invasion of heterogeneous glioma initiating cells in a microfluidic coculture platform

Chonan Yuta, Taki Sotaro, Sampetrean Oltea, Saya Hideyuki, Sudo Ryo

Integrative Biology (United Kingdom) 9 ( 9 ) 762 - 773 2017.09

ISSN 1757-9694

Glioblastoma (GBM) is a highly invasive primary brain tumor that displays cellular heterogeneity, which is composed of glioma initiating cells (GICs) and their differentiated progeny. GICs play an important role in driving aggressive invasion. In particular, the interaction between GICs and blood vessels is critical because blood vessels are known to serve as routes for the invasion of GICs. However, the effect of endothelial cells on the three-dimensional (3D) invasion process of GICs as well as the spatial relationship between GICs and their differentiated progeny remains unclear. Here, we utilized a microfluidic device to recapitulate the 3D brain tumor microenvironments constituted by human umbilical vein endothelial cells (HUVECs) and type I collagen. Using the device, we found that HUVECs promoted the 3D invasion of heterogeneous glioma cell populations into type I collagen gel. The invasion induced by HUVECs was predominantly preceded by cells positive for nestin, a neural stem cell marker. In contrast, cells positive for tubulin β3 (TUBB3), a differentiated cell marker, rarely preceded invasion. In addition, HUVECs induced the upregulation of TUBB3 in GICs. Finally, we found that the genes associated with invasion, such as integrins α2 and β3, were significantly upregulated in the presence of HUVECs. These results as well as the experimental approach provide valuable knowledge for the development of effective therapeutic strategies targeting the aggressive invasion of GBM.

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ZEB1 expression is a potential indicator of invasive endometriosis

Furuya Masataka, Masuda Hirotaka, Hara Kanako, Uchida Hiroshi, Sato Kenji, Sato Suguru, Asada Hironori, Maruyama Tetsuo, Yoshimura Yasunori, Katabuchi Hidetaka, Tanaka Mamoru, Saya Hideyuki

Acta Obstetricia et Gynecologica Scandinavica 96 ( 9 ) 1128 - 1135 2017.09

ISSN 0001-6349

Introduction: Although endometriosis is a benign disease, it shares some features with cancers, such as invasiveness and the potential to metastasize. This study sought to investigate the epithelial-mesenchymal transition status in human endometriotic lesions. Material and methods: Thirteen endometriosis patients and 10 control women without endometriosis undergoing surgery for benign indications were recruited. We examined the expression of E-cadherin, vimentin, and epithelial-mesenchymal transition-induced transcriptional factors, such as Snail and ZEB1, by immunohistochemistry. We evaluated the expression of each marker in epithelial cells of both endometriotic lesions (ovarian endometrioma, deep infiltrating endometriosis, adenomyosis) and normal endometria. The correlation between ZEB1 expression and serum level of CA125 was also investigated. Results: Immunohistochemical analysis revealed that although E-cadherin, vimentin, and Snail were expressed in epithelia of normal endometria and endometriotic lesions, ZEB1 expression was only expressed in epithelia of endometriotic lesions. Additionally, ZEB1 was most frequently observed in epithelial cells of invasive endometriosis. The endometriosis patients with high serum CA125 level were more likely to have ZEB1-positive lesions. Conclusions: This is the first observation of ZEB1 expression in epithelial cells of benign disease. The preferential expression of ZEB1 in epithelial cells of endometriotic lesions suggests that these cells may have, at least in part, a higher level of mesenchymal features possibly via ZEB1-driven epithelial-mesenchymal transition than normal endometria and that ZEB1 can be a potential indicator of invasiveness or severity of endometriosis.

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Calcitriol exerts an anti-tumor effect in osteosarcoma by inducing the endoplasmic reticulum stress response

Shimizu Takatsune, Kamel Walied A., Yamaguchi-Iwai Sayaka, Fukuchi Yumi, Muto Akihiro, Saya Hideyuki

Cancer Science 108 ( 9 ) 1793 - 1802 2017.09

ISSN 1347-9032

Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)-approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti-proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti-osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.

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Activation of Transforming Growth Factor Beta 1 Signaling in Gastric Cancer-associated Fibroblasts Increases Their Motility, via Expression of Rhomboid 5 Homolog 2, and Ability to Induce Invasiveness of Gastric Cancer Cells

Ishimoto Takatsugu, Miyake Keisuke, Nandi Tannistha, Yashiro Masakazu, Onishi Nobuyuki, Huang Kie Kyon, Lin Suling Joyce, Kalpana Ramnarayanan, Tay Su Ting, Suzuki Yuka, Cho Byoung Chul, Kuroda Daisuke, Arima Kota, Izumi Daisuke, Iwatsuki Masaaki, Baba Yoshifumi, Oki Eiji, Watanabe Masayuki, Saya Hideyuki, Hirakawa Kosei, Baba Hideo, Tan Patrick

Gastroenterology 153 ( 1 ) 191 - 204.e16 2017.07

ISSN 0016-5085

Background & Aims Fibroblasts that interact with cancer cells are called cancer-associated fibroblasts (CAFs), which promote progression of different tumor types. We investigated the characteristics and functions of CAFs in diffuse-type gastric cancers (DGCs) by analyzing features of their genome and gene expression patterns. Methods We isolated CAFs and adjacent non-cancer fibroblasts (NFs) from 110 gastric cancer (GC) tissues from patients who underwent gastrectomy in Japan from 2008 through 2016. Cells were identified using specific markers of various cell types by immunoblot and flow cytometry. We selected pairs of CAFs and NFs for whole-exome and RNA sequencing analyses, and compared expression of specific genes using quantitative reverse transcription PCR. Protein levels and phosphorylation were compared by immunoblot and immunofluorescence analyses. Rhomboid 5 homolog 2 (RHBDF2) was overexpressed from a transgene in fibroblasts or knocked down using small interfering RNAs. Motility and invasiveness of isolated fibroblasts and GC cell lines (AGS, KATOIII, MKN45, NUGC3, NUGC4, OCUM-2MD3 and OCUM-12 cell lines) were quantified by real-time imaging analyses. We analyzed 7 independent sets of DNA microarray data from patients with GC and associated expression levels of specific genes with patient survival times. Nude mice were given injections of OCUM-2MD3 in the stomach wall; tumors and metastases were collected and analyzed by immunohistochemistry. Results Many of the genes with increased expression in CAFs compared with NFs were associated with transforming growth factor beta 1 (TGFB1) activity. When CAFs were cultured in extracellular matrix, they became more motile than NFs; DGC cells incubated with CAFs were also more motile and invasive in vitro than DGC cells not incubated with CAFs. When injected into nude mice, CAF-incubated DGC cells invaded a greater number of lymphatic vessels than NF-incubated DGC cells. We identified RHBDF2 as a gene overexpressed in CAFs compared with NFs. Knockdown of RHBDF2 in CAFs reduced their elongation and motility in response to TGFB1, whereas overexpression of RHBDF2 in NFs increased their motility in extracellular matrix. RHBDF2 appeared to regulate oncogenic and non-canonical TGFB1 signaling. Knockdown of RHBDF2 in CAFs reduced cleavage of the TGFB receptor 1 (TGFBR1) by ADAM metallopeptidase domain 17 (ADAM17 or TACE) and reduced expression of genes that regulate motility. Incubation of NFs with in interleukin 1 alpha (IL1A), IL1B or tumor necrosis factor, secreted by DGCs, increased fibroblast expression of RHBDF2. Simultaneous high expression of these cytokines in GC samples was associated with shorter survival times of patients. Conclusions In CAFs isolated from human DGCs, we observed increased expression of RHBDF2, which regulates TGFB1 signaling. Expression of RHBDF2 in fibroblasts is induced by inflammatory cytokines (such as IL1A, IL1B, and tumor necrosis factor) secreted by DGCs. RHBDF2 promotes cleavage of TGFBR1 by activating TACE and motility of CAFs in response to TGFB1. These highly motile CAFs induce DGCs to invade extracellular matrix and lymphatic vessels in nude mice.

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Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression

Kasai Shin, Furuichi Yoshiyuki, Ando Norie, Kagami Keiko, Abe Masako, Nakane Takaya, Goi Kumiko, Inukai Takeshi, Saitoh Sei, Ohno Shinichi, Okazaki Shogo, Nagano Osamu, Saya Hideyuki, Sugita Kanji

Cell death & disease 8 ( 6 ) e2857 2017.06

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

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Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

Nishino Makoto, Ozaki Mari, Hegab Ahmed E., Hamamoto Junko, Kagawa Shizuko, Arai Daisuke, Yasuda Hiroyuki, Naoki Katsuhiko, Soejima Kenzo, Saya Hideyuki, Betsuyaku Tomoko

Journal of Cancer 8 ( 10 ) 1774 - 1785 2017.06

ISSN 1837-9664

Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines. Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS). Results: Both CD44v^high/ALDH^high and CD44v^high/ALDH^low cells were enriched in cells with CSC characteristics, with the CD44v^high/ALDH^low cells being more proliferative and more resistant to chemotherapeutics, whereas CD44v^high/ALDH^high cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44v^high/ALDH^low cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44v^high/ALDH^high and CD44v^high/ALDH^low cells. CD44v^high expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression. Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44v^high/ALDH^low cells and others to CD44v^high/ALDH^high cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

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Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells

Kikuchi Ryogo, Sampetrean Oltea, Saya Hideyuki, Yoshida Kazunari, Toda Masahiro

Journal of Neuro-Oncology 1 - 11 2017.05

ISSN 0167-594X

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.

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Phase 1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407)

Shitara Kohei, Doi Toshihiko, Nagano Osamu, Fukutani Miki, Hasegawa Hiromi, Nomura Shogo, Sato Akihiro, Kuwata Takeshi, Asai Kai, Einaga Yasuaki, Tsuchihashi Kenji, Suina Kentaro, Maeda Yusuke, Saya Hideyuki, Ohtsu Atsushi

Gastric Cancer 1 - 6 2017.05

ISSN 1436-3291

A previous dose-escalation study of sulfasalazine (SSZ), an inhibitor of cystine-glutamate exchange transporter xc (–), in the variant form of CD44 (CD44v)-positive cancer stem cells (CSCs) suggested that administration of SSZ induces the reduction of CD44v-positive cells and intracellular reduced glutathione (GSH) levels in patients with advanced gastric cancer (AGC). Here we report a study to evaluate SSZ in combination with cisplatin in patients with CD44v-expressing AGC refractory to cisplatin. SSZ was given by oral administration four times daily with 2 weeks on and 1 week off. Cisplatin at 60 mg/m² was administered every 3 weeks. Of the 15 patients who underwent prescreening of CD44v expression, 8 patients were positive, and 7 patients were treated with the dose level of SSZ at 6 g/day. One patient experienced dose-limiting toxicity (DLT) as grade 3 anorexia. Although no other patients experienced DLT, 4 patients required dose interruption or reduction of SSZ; thus, we terminated further dose escalation. No patient achieved objective response, but 1 patient completed six cycles with stable disease for more than 4 months as well as reduction of intratumoral GSH level. The combination of SSZ plus cisplatin was manageable, although dose modification was frequently required during a short observational period.

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Rheostatic CD44 isoform expression and its association with oxidative stress in human malignant mesothelioma

Chew Shan Hwu, Okazaki Yasumasa, Akatsuka Shinya, Wang Shenqi, Jiang Li, Ohara Yuuki, Ito Fumiya, Saya Hideyuki, Sekido Yoshitaka, Toyokuni Shinya

Free Radical Biology and Medicine 106 91 - 99 2017.05

ISSN 0891-5849

CD44 exists as a standard (CD44s) isoform and different variant isoforms (CD44v) due to alternative splicing. While the complex nature of these different isoforms has not been fully elucidated, CD44v expression has been shown to exert oncogenic effects by promoting tumor progression, metastasis and resistance of tumor cells to chemotherapy. One of the CD44v isoforms, CD44v8-10, was recently shown to protect cancer cells from oxidative stress by increasing the synthesis of glutathione (GSH). However, data regarding CD44 isoform expression in malignant mesothelioma (MM) are still lacking. Here, we show that most of the MM cell lines express both the CD44s and CD44v isoforms, in contrast to non-tumorigenic mesothelial cells, which express only CD44s. Moreover, we show here that these MM cell lines are positive for CD44 variable exon 9, with CD44v8-10 among the variant isoforms expressed. The expression of CD44 variable exon 9 was found to be statistically associated with NF2 inactivation, a common occurrence in MM. Knockdown of CD44 reduced the protein level of xCT, a cystine transporter, and increased oxidative stress. However, an increase in GSH was also observed and was associated with enhanced chemoresistance in CD44-knockdown cells. Increased GSH was mediated by the Nrf2/AP-1-induced upregulation of GCLC, a subunit of the enzyme catalyzing GSH synthesis. Our results thus suggest that the response to CD44 depletion is cell type-dependent and, in cases such as MM cells, compensatory pathway(s) might be activated rheostatically to account for the loss of CD44 and counteract enhanced oxidative stress.

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FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia

Ishizawa Jo, Sugihara Eiji, Kuninaka Shinji, Mogushi Kaoru, Kojima Kensuke, Benton Christopher B., Zhao Ran, Chachad Dhruv, Hashimoto Norisato, Jacamo Rodrigo O., Qiu Yihua, Yoo Suk Young, Okamoto Shinichiro, Andreeff Michael, Kornblau Steven M., Saya Hideyuki

Blood 129 ( 14 ) 1958 - 1968 2017.04

ISSN 0006-4971

FZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G₁ phase, and activation of the G₂-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage–induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

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Blocking COX-2 induces apoptosis and inhibits cell proliferation via the Akt/survivin- and Akt/ID3 pathway in low-grade-glioma

Sato Aya, Mizobuchi Yoshifumi, Nakajima Kohei, Shono Kenji, Fujihara Toshitaka, Kageji Teruyoshi, Kitazato Keiko, Matsuzaki Kazuhito, Mure Hideo, Kuwayama Kazuyuki, Sumi Akiko, Saya Hideyuki, Sampetrean Oltea, Nagahirao Shinji

Journal of Neuro-Oncology 132 ( 2 ) 231 - 238 2017.04

ISSN 0167-594X

Approximately half of surgically-treated patients with low-grade-glioma (LGG) suffer recurrence or metastasis. Currently there is no effective drug treatment. While the selective COX-2 inhibitor celecoxib showed anti-neoplastic activity against several malignant tumors, its effects against LGG remain to be elucidated. Ours is the first report that the expression level of COX-2 in brain tissue samples from patients with LGG and in LGG cell lines is higher than in the non-neoplastic region and in normal brain cells. We found that celecoxib attenuated LGG cell proliferation in a dose-dependent manner. It inhibited the generation of prostaglandin E2 and induced apoptosis and cell-cycle arrest. We also show that celecoxib hampered the activation of the Akt/survivin- and the Akt/ID3 pathway in LGGs. These findings suggest that celecoxib may have a promising therapeutic potential and that the early treatment of LGG patients with the drug may be beneficial.

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