Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Sulfasalazine, a therapeutic agent for ulcerative colitis, inhibits the growth of CD44v9⁺ cancer stem cells in ulcerative colitis-related cancer

Seishima Ryo, Okabayashi Koji, Nagano Osamu, Hasegawa Hirotoshi, Tsuruta Masashi, Shimoda Masayuki, Kameyama Kaori, Saya Hideyuki, Kitagawa Yuko

Clinics and Research in Hepatology and Gastroenterology 2015

ISSN 2210-7401

Background and objective: Sulfasalazine reduces the risk of ulcerative colitis (UC)-related cancer through its anti-inflammatory effect and induction of oxidative stress in cancer cells by inhibiting the glutamate-cystine transporter, which is closely associated with the cancer stem cell surface marker CD44v9. This study aimed to quantify the effects of sulfasalazine on CD44v9 expression and pathological factors in colorectal cancers (CRCs) arising from UC. Methods: Twenty-six patients with UC-related cancer were classified into groups according to the length of sulfasalazine treatment as follows: (1) long-term (LT) (≥ 5 years) and (2) short-term (ST) (<5 years). Using immunohistochemistry, we compared CD44v9 and Ki-67 expression and pathological characteristics of each group's tumors. In vitro assay was performed to investigate the effect of sulfasalazine on epithelial differentiation and proliferation of CD44⁺ cancer cells. Results: Immunohistochemical analysis revealed that CD44v9 expression tended to be lower in the LT group (LT:ST=15.4%:46.2%, P =0.20), and Ki-67/CD44v9 double-stained cells were significantly lower in the LT group (LT:ST=0%:6.9%, P =0.01). Pathologically, the frequency of well-differentiated adenocarcinomas was higher in the LT group (LT:ST=84.6%:38.5%, P =0.04). In vitro assay revealed that sulfasalazine promoted the expression of epithelial differentiation markers (E-cadherin and CDX2) and inhibited the proliferation of CD44⁺ cancer cells. Conclusions: Long-term sulfasalazine administration reduced proliferative CD44v9⁺ cells and increased the degree of differentiation of adenocarcinomas. These findings indicate the importance of CD44v9⁺ cells in UC-related cancer progression and suggest that sulfasalazine may serve as a novel therapeutic agent that targets CD44v9⁺ cells.

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Salt-Inducible Kinase 2 Couples Ovarian Cancer Cell Metabolism with Survival at the Adipocyte-Rich Metastatic Niche

Miranda Fabrizio, Mannion David, Liu Shujuan, Zheng Yiyan, Mangala Lingegowda S., Redondo Clara, Herrero-Gonzalez Sandra, Xu Ruoyan, Taylor Charlotte, Chedom Donatien Fotso, Karaminejadranjbar Mohammad, Albukhari Ashwag, Jiang Dahai, Pradeep Sunila, Rodriguez-Aguayo Cristian, Lopez-Berestein Gabriel, Salah Eidarus, Abdul Azeez Kamal R., Elkins Jonathan M., Campo Leticia, Myers Kevin A., Klotz Daniel, Bivona Serena, Dhar Sunanda, Bast Robert C., Saya Hideyuki, Choi Hwan Geun, Gray Nathanael S., Fischer Roman, Kessler Benedikt M., Yau Christopher, Sood Anil K., Motohara Takeshi, Knapp Stefan, Ahmed Ahmed Ashour

Cancer Cell 2014.12

ISSN 1535-6108

The adipocyte-rich microenvironment forms a niche for ovarian cancer metastasis, but the mechanisms driving this process are incompletely understood. Here we show that salt-inducible kinase 2 (SIK2) is overexpressed in adipocyte-rich metastatic deposits compared with ovarian primary lesions. Overexpression of SIK2 in ovarian cancer cells promotes abdominal metastasis while . SIK2 depletion prevents metastasis in vivo. Importantly, adipocytes induce calcium-dependent activation and autophosphorylation of SIK2. Activated SIK2 plays a dual role in augmenting AMPK-induced phosphorylation of acetyl-CoA carboxylase and in activating the PI3K/AKT pathway through p85α-S154 phosphorylation. These findings identify SIK2 at the apex of the adipocyte-induced signaling cascades in cancer cells and make a compelling case for targeting SIK2 for therapy in ovarian cancer. Miranda et al. show that in serous ovarian cancer SIK2 is overexpressed in adipocyte-rich metastatic deposits compared with primary lesions. Adipocytes induce calcium-dependent activation and autophosphorylation of SIK2, which drives tumor cell survival and metabolism. SIK2 depletion prevents metastasis in vivo.

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Functional analysis of a novel glioma antigen, EFTUD1

Saito Katsuya, Iizuka Yukihiko, Ohta Shigeki, Takahashi Satoshi, Nakamura Kenta, Saya Hideyuki, Yoshida Kazunari, Kawakami Yutaka, Toda Masahiro

Neuro-Oncology 16 ( 12 ) 1618 - 1629 2014.12

ISSN 1522-8517

BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.

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IGF2 preserves osteosarcoma cell survival by creating an autophagic state of dormancy that protects cells against chemotherapeutic stress

Shimizu Takatsune, Sugihara Eiji, Yamaguchi-Iwai Sayaka, Tamaki Sakura, Koyama Yuko, Kamel Walied, Ueki Arisa, Ishikawa Tomoki, Chiyoda Tatsuyuki, Osuka Satoru, Onishi Nobuyuki, Ikeda Hiroko, Kamei Junzo, Matsuo Koichi, Fukuchi Yumi, Nagai Toshihiro, Toguchida Junya, Toyama Yoshiaki, Muto Akihiro, Saya Hideyuki

Cancer Research 74 ( 22 ) 6531 - 6541 2014.11

ISSN 0008-5472

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma.

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The use of next-generation sequencing in molecular diagnosis of neurofibromatosis type 1

Maruoka Ryo, Takenouchi Toshiki, Torii Chiharu, Shimizu Atsushi, Misu Kumiko, Higasa Koichiro, Matsuda Fumihiko, Ota Arihito, Tanito Katsumi, Kuramochi Akira, Arima Yoshimi, Otsuka Fujio, Yoshida Yuichi, Moriyama Keiji, Niimura Michihito, Saya Hideyuki, Kosaki Kenjiro

Genetic Testing and Molecular Biomarkers 18 ( 11 ) 722 - 735 2014.11

AIMS: We assessed the validity of a next-generation sequencing protocol using in-solution hybridization-based enrichment to identify NF1 mutations for the diagnosis of 86 patients with a prototypic genetic syndrome, neurofibromatosis type 1. In addition, other causative genes for classic genetic syndromes were set as the target genes for coverage analysis.RESULTS: The protocol identified 30 nonsense, 19 frameshift, and 8 splice-site mutations, together with 10 nucleotide substitutions that were previously reported to be pathogenic. In the remaining 19 samples, 10 had single-exon or multiple-exon deletions detected by a multiplex ligation-dependent probe amplification method and 3 had missense mutations that were not observed in the normal Japanese SNP database and were predicted to be pathogenic. Coverage analysis of the genes other than the NF1 gene included on the same diagnostic panel indicated that the mean coverage was 115-fold, a sufficient depth for mutation detection.CONCLUSIONS: The overall mutation detection rate using the currently reported method in 86 patients who met the clinical diagnostic criteria was 92.1% (70/76) when 10 patients with large deletions were excluded. The results validate the clinical utility of this next-generation sequencing-based method for the diagnosis of neurofibromatosis type 1. Comparable detection rates can be expected for other genetic syndromes, based on the results of the coverage analysis.

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A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

Watanabe Yusaku, Yoshimura Kiyoshi, Yoshikawa Koichi, Tsunedomi Ryoichi, Shindo Yoshitaro, Matsukuma Sou, Maeda Noriko, Kanekiyo Shinsuke, Suzuki Nobuaki, Kuramasu Atsuo, Sonoda Kouhei, Tamada Koji, Kobayashi Sei, Saya Hideyuki, Hazama Shoichi, Oka Masaki

International Journal of Oncology 45 ( 5 ) 1857 - 1866 2014.11

ISSN 1019-6439

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

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Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis

Techasen Anchalee, Loilome Watcharin, Namwat Nisana, Khuntikeo Narong, Puapairoj Anucha, Jearanaikoon Patcharee, Saya Hideyuki, Yongvanit Puangrat

Tumor Biology 35 ( 9 ) 8645 - 8652 2014.10

ISSN 1010-4283

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT) in tumors. In this study, we investigated the expression of E-cadherin and its association with cancer invasion and prognosis in cholangiocarcinoma (CCA). Immunohistochemistry results demonstrated a statistically significant association between the positive metastasis status with low E-cadherin protein expression in human CCA tissues (P = 0.04). Statistical trends were identified for low E-cadherin level and shorter survival time (P = 0.08). Targeting the E-cadherin expression in CCA cells with siRNA caused upregulation of vimentin, a mesenchymal marker, and disappearance of the E-cadherin/β-catenin adhesion complex from cell membranes. Moreover, migration and invasion abilities of the cells were increased under this condition. These findings suggest that reduction of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, which affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis. Thus, E-cadherin can act as a central modulator of tumor cell phenotype and is a potential metastasis marker in CCA.

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TNFα promotes osteosarcoma progression by maintaining tumor cells in an undifferentiated state

Mori T., Sato Y., Miyamoto K., Kobayashi T., Shimizu T., Kanagawa H., Katsuyama E., Fujie A., Hao W., Tando T., Iwasaki R., Kawana H., Morioka H., Matsumoto M., Saya H., Toyama Y., Miyamoto T.

Oncogene 33 ( 33 ) 4236 - 4241 2014.08

ISSN 0950-9232

Chronic inflammation is frequently associated with tumorigenesis in elderly people. By contrast, young people without chronic inflammation often develop tumors considered independent of chronic inflammation but driven instead by mutations. Thus, whether inflammation has a significant role in tumor progression in tumors driven by mutations remains largely unknown. Here we show that TNFα is required for the tumorigenesis of osteosarcoma, the most common tumor in children and adolescents. We show that transplantation of AX osteosarcoma cells, which harbor mutations driving c-Myc overexpression and Ink4a-deficiency, in wild-type mice promotes lethal tumorigenesis accompanied by ectopic bone formation and multiple metastases, phenotypes seen in osteosarcoma patients. Such tumorigenesis was completely abrogated in TNFα-deficient mice. AX cells have the capacity to undergo osteoblastic differentiation; however, that activity was significantly inhibited by TNFα treatment, suggesting that TNFα maintains AX cells in an undifferentiated state. TNFα inhibition of AX cell osteoblastic differentiation occurred through ERK activation, and a pharmacological TNFα inhibitor effectively inhibited both AX cell tumorigenesis and increased osteoblastic gene expression and increased survival of tumor-bearing mice. Lethal tumorigenesis of AX cells was also abrogated in IL-1α/IL-1β doubly deficient mice. We found that both TNFα and IL-1 maintained AX cells in an undifferentiated state via ERK activation. Thus, inflammatory cytokines are required to promote tumorigenesis even in mutation-induced tumors, and TNFα/IL-1 and ERK may represent therapeutic targets for osteosarcoma.

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TNF-/TNFR1 signaling promotes gastric tumorigenesis through induction of Noxo1 and Gna14 in tumor cells

Oshima H., Ishikawa T., Yoshida G. J., Naoi K., Maeda Y., Naka K., Ju X., Yamada Y., Minamoto T., Mukaida N., Saya H., Oshima M.

Oncogene 33 ( 29 ) 3820 - 3829 2014.07

ISSN 0950-9232

Helicobacter pylori infection induces chronic inflammation that contributes to gastric tumorigenesis. Tumor necrosis factor (TNF-) is a proinflammatory cytokine, and polymorphism in the TNF- gene increases the risk of gastric cancer. We herein investigated the role of TNF- in gastric tumorigenesis using Gan mouse model, which recapitulates human gastric cancer development. We crossed Gan mice with TNF- (Tnf) or TNF- receptor TNFR1 (Tnfrsf1a) knockout mice to generate Tnf-/-Gan and Tnfrsf1a-/-Gan mice, respectively, and examined their tumor phenotypes. Notably, both Tnf-/-Gan mice and Tnfrsf1a-/-Gan mice showed similar, significant suppression of gastric tumor growth compared with control Tnf+/+ or Tnfrsf1a+/+ Gan mice. These results indicate that TNF- signaling through TNFR1 is important for gastric tumor development. Bone marrow (BM) transplantation experiments showed that TNF- expressed by BM-derived cells (BMDCs) stimulates the TNFR1 on BMDCs by an autocrine or paracrine manner, which is important for gastric tumor promotion. Moreover, the microarray analysis and colony formation assay indicated that NADPH oxidase organizer 1 (Noxo1) and Gna14 are induced in tumor epithelial cells in a TNF-dependent manner, and have an important role in tumorigenicity and tumor-initiating cell property of gastric cancer cells. Accordingly, it is possible that the activation of TNF-/TNFR1 signaling in the tumor microenvironment promotes gastric tumor development through induction of Noxo1 and Gna14, which contribute to maintaining the tumor cells in an undifferentiated state. The present results indicate that targeting the TNF-/TNFR1 pathway may be an effective preventive or therapeutic strategy for gastric cancer.

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Effectiveness of plasma treatment on gastric cancer cells

Torii Koji, Yamada Suguru, Nakamura Kae, Tanaka Hiromasa, Kajiyama Hiroaki, Tanahashi Kuniaki, Iwata Naoki, Kanda Mitsuro, Kobayashi Daisuke, Tanaka Chie, Fujii Tsutomu, Nakayama Goro, Koike Masahiko, Sugimoto Hiroyuki, Nomoto Shuji, Natsume Atsushi, Fujiwara Michitaka, Mizuno Masaaki, Hori Masaru, Saya Hideyuki, Kodera Yasuhiro

Gastric Cancer 18 ( 3 ) 635 - 643 2014.07

ISSN 1436-3291

Background: Treatment of peritoneal carcinomatosis arising from gastric cancer remains a considerable challenge. In recent years, the anticancer effect of nonequilibrium atmospheric pressure plasma (NEAPP) has been reported in several cancer cell lines. Use of NEAPP may develop into a new class of anticancer therapy that augments surgery, chemotherapy, and radiotherapy. Method: Gastric cancer cells were assessed for changes in cell morphology and rate of proliferation after treatment with NEAPP-exposed medium (PAM). To explore the functional mechanism, caspase 3/7, annexin V, and uptake of reactive oxygen species (ROS) were evaluated, along with the effect of the ROS scavenger N-acetylcysteine (NAC). Results: PAM treatment for 24 h affected cell morphology, suggestive of induction of apoptosis. PAM cytotoxicity was influenced by the time of exposure to PAM, the type of cell line, and the number of cells seeded. Cells treated with PAM for 2 h demonstrated activated caspase 3/7 and an increased proportion of annexin V-positive cells compared with untreated cells. Additionally, ROS uptake was observed in PAM-treated cells, whereas NAC reduced the cytotoxicity induced by PAM presumably through reduction of ROS uptake. Furthermore, CD44 variant 9, which reportedly leads to glutathione synthesis and suppresses stress signaling of ROS, was overexpressed in PAM-resistant cells. Conclusions: PAM treatment induced apoptosis of gastric cancer cells through generation and uptake of ROS. Local administration of PAM could develop into an option to treat peritoneal carcinomatosis.

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TSPAN2 Is Involved in Cell Invasion and Motility during Lung Cancer Progression

Otsubo Chihiro, Otomo Ryo, Miyazaki Makoto, Matsushima-Hibiya Yuko, Kohno Takashi, Iwakawa Reika, Takeshita Fumitaka, Okayama Hirokazu, Ichikawa Hitoshi, Saya Hideyuki, Kiyono Tohru, Ochiya Takahiro, Tashiro Fumio, Nakagama Hitoshi, Yokota Jun, Enari Masato

Cell Reports 7 ( 2 ) 527 - 538 2014.04

In lung cancer progression, p53 mutations are more often observed in invasive tumors than in noninvasive tumors, suggesting that p53 is involved in tumor invasion and metastasis. To understand the nature of p53 function as a tumor suppressor, it is crucial to elucidate the detailed mechanism of the alteration in epithelial cells that follow oncogenic KRAS activation and p53 inactivation. Here, we report that KRAS activation induces epithelial-mesenchymal transitionand that p53 inactivation is required for cell motility and invasiveness. Furthermore, TSPAN2, atransmembrane protein, is responsible for cell motility and invasiveness elicited by p53 inactivation. TSPAN2 is highly expressed in p53-mutated lung cancer cells, and high expression of TSPAN2 is associated with the poor prognosis of lung adenocarinomas. TSPAN2 knockdown suppresses metastasis tothe lungs and liver, enabling prolonged survival. TSPAN2 enhances cell motility and invasiveness by assisting CD44 in scavenging intracellular reactive oxygen species.

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Targeting Cancer Stem Cells-Modulating Embryonic Stem Cell Signaling, Epigenetics, and Tumor Metabolism

Takebe Naoko, Harris Pamela Jo, Kondo Yutaka, Nair Abhilasha, Percy Ivy S., Saya Hideyuki

295 - 317 2014.04

Cancer stem cells (CSC) are important to understand in order to develop solutions to treat tumor metastasis and resistance to therapy. CSCs are characterized by their ability to self-renew and differentiate into tumors, ultimately leading to therapeutic resistance. This chapter summarizes the various potential therapeutic approaches to control CSCs survival and proliferation based on preclinical and clinical study-based rationale. The proposed agents range from investigational, which are still in pre-clinical stage, already in early clinical investigational stage, or already approved for clinical use for other indications. The manuscript covers the agents targeting embryonic stem cell signaling, epigenetic control, cancer metabolism, and induction of epithelial-to-mesenchymal transition (EMT). The combination of therapeutic strategies to reduce bulk tumor populations and to target CSCs population may provide clinically meaningful tumor suppression. The development of new anti-CSC therapeutic agents and the complexity of potential molecular pathways controlling CSC survival are described in this review.

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Glycan profiling of gestational choriocarcinoma using a lectin microarray

Kobayashi Yusuke, Masuda Kenta, Banno Kouji, Kobayashi Nana, Umene Kiyoko, Nogami Yuya, Tsuji Kosuke, Ueki Arisa, Nomura Hiroyuki, Sato Kenji, Tominaga Eiichiro, Shimizu Takatsune, Saya Hideyuki, Aoki Daisuke

Oncology Reports 31 ( 3 ) 1121 - 1126 2014.03

ISSN 1021-335X

Glycosylation is an important post-translational modification, in which attachment of glycans to proteins has effects on biological functions and carcinogenesis. Analysis of human chorionic gonadotropin, a glycoprotein hormone produced by placental trophoblasts and trophoblastic tumors, has contributed to the diagnosis and treatment of trophoblastic disease, resulting in reduced incidence and mortality. However, alterations of the glycan structure itself in choriocarcinoma have not been characterized. We established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC³-1), which mimics the clinical pathohistology in vivo, to examine the tumorigenesis and pathogenesis of choriocarcinoma. In this study, the alterations of glycan structures in the development of choriocarcinoma were examined by performance of comprehensive glycan profiling in clinical samples and in iC³-1 cells using a conventional microarray and the recently introduced lectin microarray. Microarray comparison showed significant upregulation of several characteristic glycogenes in the iC³-1 cells as compared to the parental HTR8/SVneo cells. The lectin array showed increased α-2-6-sialic acid, Galβ1-4GlcNAc, GlcNAcβ1-3GalNAc, and decreased α-1-6 core fucose, high mannose, GalNacβ1-4Gal, GALNAc (Tn antigen) and Galβ-3Gal in choriocarcinoma tissue compared to normal villi. This is the first report of a lectin array analysis in choriocarcinoma and provides useful information for understanding of the disease.

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CD44s signals the acquisition of the mesenchymal phenotype required for anchorage-independent cell survival in hepatocellular carcinoma

Okabe H., Ishimoto T., Mima K., Nakagawa S., Hayashi H., Kuroki H., Imai K., Nitta H., Saito S., Hashimoto D., Chikamoto A., Ishiko T., Watanabe M., Nagano O., Beppu T., Saya H., Baba H.

British Journal of Cancer 110 ( 4 ) 958 - 966 2014.02

ISSN 0007-0920

Background:Circulating tumour cells (CTCs) have an important role in metastatic processes, but details of their basic characteristics remain elusive. We hypothesised that CD44-expressing CTCs show a mesenchymal phenotype and high potential for survival in hepatocellular carcinoma (HCC).Methods:Circulating CD44 + CD90 + cells, previously shown to be tumour-initiating cells, were sorted from human blood and their genetic characteristics were compared with those of tumour cells from primary tissues. The mechanism underlying the high survival potential of CD44-expressing cells in the circulatory system was investigated in vitro.Results:CD44 + CD90 + cells in the blood acquired epithelial-mesenchymal transition, and CD44 expression remarkably increased from the tissue to the blood. In Li7 and HLE cells, the CD44 high population showed higher anoikis resistance and sphere-forming ability than did the CD44 low population. This difference was found to be attributed to the upregulation of Twist1 and Akt signal in the CD44 high population. Twist1 knockdown showed remarkable reduction in anoikis resistance, sphere formation, and Akt signal in HLE cells. In addition, mesenchymal markers and CD44s expression were downregulated in the Twist1 knockdown.Conclusions:CD44s symbolises the acquisition of a mesenchymal phenotype regulating anchorage-independent capacity. CD44s-expressing tumour cells in peripheral blood are clinically important therapeutic targets in HCC.

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Multiple café au lait spots in familial patients with MAP2K2 mutation

Takenouchi Toshiki, Shimizu Atsushi, Torii Chiharu, Kosaki Rika, Takahashi Takao, Saya Hideyuki, Kosaki Kenjiro

American Journal of Medical Genetics, Part A 164 ( 2 ) 392 - 396 2014.02

ISSN 1552-4825

Recent advances in genetic diagnostic technologies have made the classic disease nosology highly complicated. This situation is exemplified by rasopathies, among which neurofibromatosis type 1 and Noonan syndrome represent prototypic entities. The former condition is characterized by multiple café au lait spots and neurofibromas, while the latter is characterized by distinct facial features, webbed neck, congenital heart disease, and a short stature. On rare occasions, the features of both neurofibromatosis and Noonan syndrome co-exist within an individual; such patients are diagnosed as having neurofibromatosis-Noonan syndrome. Here, we report familial patients with multiple café au lait spots and Noonan syndrome-like facial features. A mutation analysis unexpectedly revealed a mutation in MAP2K2 in both the propositus and his mother. The propositus fulfilled the diagnostic criteria for neurofibromatosis type 1, but his mother did not. Their phenotype was not consistent with that of cardio-facio-cutaneous syndrome, which is classically known to be associated with MAP2K2 mutations. The mother of the propositus had cervical cancer at the age of 23 years, consistent with the oncogenic tendency associated with rasopathies. The phenotypic combination of multiple café au lait spots and Noonan syndrome-like facial features suggested a diagnosis of neurofibromatosis-Noonan syndrome. Whether this condition represents a discrete disease entity or a variable expression of neurofibromatosis type 1 has long been debated. The present observation suggests that some perturbation in the RAS/MAPK signaling cascade results in multiple café au lait spots, a key diagnostic phenotype of rasopathies, although the exact mechanism remains to be elucidated.

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Ink4a/Arf^-/- and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f^- quiescent cells

Kai K., Iwamoto T., Kobayashi T., Arima Y., Takamoto Y., Ohnishi N., Bartholomeusz C., Horii R., Akiyama F., Hortobagyi G. N., Pusztai L., Saya H., Ueno N. T.

Oncogene 33 ( 4 ) 440 - 448 2014.01

ISSN 0950-9232

Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (-/-) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf^-/- mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (TNBC) (for example, pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f^- quiescent cells and CD49f ⁺ cells. Contrary to our expectation, CD49f^- quiescent cells had high tumor-initiating potential and CD49f⁺ cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f^- quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human TNBC.

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Inversed relationship between CD44 variant and c-Myc due to oxidative stress-induced canonical Wnt activation

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 443 ( 2 ) 622 - 627 2014.01

ISSN 0006-291X

Cancer stem-like cells express high amount of CD44 variant8-10 which protects cancer cells from redox stress. We have demonstrated by immunohistochemical analysis and Western blotting, and reverse-transcription polymerase chain reaction, that CD44 variant8-10 and c-Myc tend to show the inversed expression manner in gastric cancer cells. That is attributable to the oxidative stress-induced canonical Wnt activation, and furthermore, the up-regulation of the downstream molecules, one of which is oncogenic c-Myc, is not easily to occur in CD44 variant-positive cancer cells. We have also found out that CD44v8-10 expression is associated with the turn-over of the c-Myc with the experiments using gastric cancer cell lines. This cannot be simply explained by the model of oxidative stress-induced Wnt activation. CD44v8-10-positive cancer cells are enriched at the invasive front. Tumor tissue at the invasive area is considered to be composed of heterogeneous cellular population; dormant cancer stem-like cells with CD44v8-10 ^high/ Fbw7 ^high/ c-Myc ^low and proliferative cancer stem-like cells with CD44v8-10 ^high/ Fbw7 ^low/ c-Myc ^high.

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EpCAM expression in the prostate cancer makes the difference in the response to growth factors

Yoshida Go J., Saya Hideyuki

Biochemical and Biophysical Research Communications 443 ( 1 ) 239 - 245 2014.01

ISSN 0006-291X

Introduction: Epithelial cell adhesion molecule (EpCAM) is expressed in tumors with an epithelial cell of origin, in a heterogeneous manner. Prostate cancer stem-like cells highly express EpCAM. However, little is known about how EpCAM is involved in the ability of cells to adapt to micro-environmental changes in available growth factors, which is one of the essential biological phenotypes of cancer stem-like cells (CSCs). Methods: EpCAM-high and EpCAM-low subpopulations of cells were established from the prostate cancer cell line PC-3. Signal transductions in response to serum starvation, and on the exposure to EGF ligand or the specific inhibitor were analyzed in terms. Furthermore, we analyzed the expression level of amino acid transporters which contribute to the activation of mTOR signal between the two subgroups. Results: EpCAM-high and EpCAM-low PC-3 subpopulations showed markedly different responses to serum starvation. EpCAM expression was positively correlated with activation of the mTOR and epithelial growth factor receptor (EGFR) signaling pathways. Furthermore, AMP-activated protein kinase (AMPK) was gradually de-activated in EpCAM-low PC-3 cells in the absence of serum. Conclusions: EpCAM regulates the AMPK signaling pathway, essential for the response to growth factors characterized by EGF. LAT1, the amino acid transporter stabilized at the cellular membrane by EpCAM, is likely to be responsible for the difference in the susceptibility to EGF between EpCAM-high and EpCAM-low PC-3 cells.

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Expression of CD24 is associated with HER2 expression and supports HER2-Akt signaling in HER2-positive breast cancer cells

Hosonaga Mari, Arima Yoshimi, Sugihara Eiji, Kohno Norio, Saya Hideyuki

Cancer Science 105 ( 7 ) 779 - 787 2014

ISSN 1347-9032

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is treated with HER2-targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3-kinase (PI3K)-Akt and MAPK pathways. However, resistance to HER2-targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2-positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple-negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24-positive cells was markedly higher in the HER2-positive fraction than in the HER2-negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2-positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K-Akt signaling. Furthermore, CD24 may contribute to resistance to HER2-targeted therapy and, therefore, is itself a potential therapeutic target in HER2-positive breast cancer. The knockdown of CD24 decreased the phosphorylation of Akt in HER2-positive breast cancer cells. These results suggest that CD24 supports HER2-Akt signaling pathway.

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Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in cancer cells and promote redox adaptation

Ishimoto Takatsugu, Sugihara Hidetaka, Watanabe Masayuki, Sawayama Hiroshi, Iwatsuki Masaaki, Baba Yoshifumi, Okabe Hirohisa, Hidaka Kosei, Yokoyama Naomi, Miyake Keisuke, Yoshikawa Momoko, Nagano Osamu, Komohara Yoshihiro, Takeya Motohiro, Saya Hideyuki, Baba Hideo

Carcinogenesis 35 ( 5 ) 1003 - 1011 2014

ISSN 0143-3334

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

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