Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells

Ohkawa Mayumi, Ohno Yoshiya, Masuko Kazue, Takeuchi Akiko, Suda Kentaro, Kubo Akihiro, Kawahara Rieko, Okazaki Shogo, Tanaka Toshiyuki, Saya Hideyuki, Seki Masayuki, Enomoto Takemi, Yagi Hideki, Hashimoto Yoshiyuki, Masuko Takashi

Biochemical and Biophysical Research Communications 406 ( 4 ) 649 - 655 2011.03

ISSN 0006-291X

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4kb. We established five homozygous LAT1-disrupted (LAT1^-/-) cell clones, derived from a heterozygous LAT1^+/- clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1^-/- DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1^-/- cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1^-/- DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1^-/- DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1^+/- DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

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CD44 Variant Regulates Redox Status in Cancer Cells by Stabilizing the xCT Subunit of System xc- and Thereby Promotes Tumor Growth

Ishimoto Takatsugu, Nagano Osamu, Yae Toshifumi, Tamada Mayumi, Motohara Takeshi, Oshima Hiroko, Oshima Masanobu, Ikeda Tatsuya, Asaba Rika, Yagi Hideki, Masuko Takashi, Shimizu Takatsune, Ishikawa Tomoki, Kai Kazuharu, Takahashi Eri, Imamura Yu, Baba Yoshifumi, Ohmura Mitsuyo, Suematsu Makoto, Baba Hideo, Saya Hideyuki

Cancer Cell 19 ( 3 ) 387 - 400 2011.03

ISSN 1535-6108

CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38 ^MAPK, a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21 ^CIP1/WAF1. These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

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[Encounter of cancer cells with bone. The significance of cancer stem cells and epithelial-mesenchymal transition in tumor invasion and metastasis].

Yoshida G. J., Saya Hideyuki

Clinical calcium 21 ( 3 ) 411 - 417 2011.03

ISSN 0917-5857

Cancer stem cells (CSCs) , which are subset of tumor cells resistant to radiation and chemotherapy, are associated with malignant characteristics of tumor and possess both self-renewal ability and pluripotency for tumor formation. In the process of generating non-CSCs from CSCs, gene mutations and epigenetic changes are induced in those cells, resulting in composition of tumor tissue with heterogeneous cell population. CSCs have been recognized as the source of metastatic foci. Epithelial-mesenchymal transition (EMT) is a change in cellular phenotype characterized by the loss of cell-to-cell adhesions and the gain of migratory behaviors,which has been shown to be a critical factor for initiating cancer invasion and metastasis. However, some recent studies suggest that EMT is not essential requirement for tumor invasion and metastasis. Herein, we discuss the biological significance of CSCs and EMT in tumor invasion and metastasis.

NKX2.2 suppresses self-renewal of glioma-initiating cells

Muraguchi Teruyuki, Tanaka Shingo, Yamada Daisuke, Tamase Akira, Nakada Mitsutoshi, Nakamura Hideo, Hoshii Takayuki, Ooshio Takako, Tadokoro Yuko, Naka Kazuhito, Ino Yasushi, Todo Tomoki, Kuratsu Jun Ichi, Saya Hideyuki, Hamada Jun Ichiro, Hirao Atsushi

Cancer Research 71 ( 3 ) 1135 - 1145 2011.02

ISSN 0008-5472

Glioblastoma (GBM) is the most aggressive and destructive form of brain cancer. Animal models that can unravel the mechanisms underlying its progression are needed to develop rational and effective molecular therapeutic approaches. In this study, we report the development of mouse models for spontaneous gliomas representing distinct progressive stages of disease that are governed by defined genetic alterations. Neural stem/ progenitor cell (NPC)-specific constitutive Ras activation in vivo plus p53 deficiency led to development of primarily anaplastic astrocytoma (grade III), whereas combined loss of p53 plus p16^Ink4a/p19^Arf led to development of GBM (grade IV) at 100% penetrance within 6 weeks. These glioma models showed enhanced stem cell properties (stemness) accompanied by malignant progression. Notably, we determined that, in our models and in human specimens, downregulation of the homeodomain transcription factor NKX2.2, which is essential for oligodendroglial differentiation, was correlated with increased tumor malignancy. NKX2.2 overexpression by GBM-derived glioma-initiating cells (GIC) induced oligodendroglial differentiation and suppressed self-renewal capacity. By contrast, Nkx2.2 downregulation in mouse NPCs accelerated GBM formation. Importantly, the inhibitory effects of NXK2.2 on GIC self-renewal were conserved in human cells. Thus, our mouse models offer pathobiologically significant advantages to investigate the nature of brain tumors, with improved opportunities to develop novel mechanism-based therapeutic approaches.

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Mad2 inhibits the mitotic kinesin MKlp2

Lee Sang Hyun, McCormick Frank, Saya Hideyuki

Journal of Cell Biology 191 ( 6 ) 1069 - 1077 2010.12

ISSN 0021-9525

We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis.

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Identification of CD44 as a cell surface marker for Müller glia precursor cells

Shinoe Toru, Kuribayashi Hiroshi, Saya Hideyuki, Seiki Motoharu, Aburatani Hiroyuki, Watanabe Sumiko

Journal of Neurochemistry 115 ( 6 ) 1633 - 1642 2010.12

ISSN 0022-3042

In the retina, both neurons and glia differentiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid receptor expressed on mature Müller glial cells. We found that in the developing mouse retina, expression of CD44 was transiently observed at or around birth in a subpopulation of c-kit-positive retinal progenitor cells. During in vitro culture, purified CD44/c-kit-positive retinal progenitor cells exclusively differentiated into Müller glial cells and not into neurons, suggesting that CD44 marks a subpopulation of retinal progenitor cells that are fated to become glia. Over-expression of CD44 inhibited the extension of processes by Müller glial cells and neurons. Notch signaling is known to be involved in the specification of retinal progenitors into a glial fate. Activation of Notch signaling increased the number of CD44-positive cells, and treatment with the Notch signal inhibitor, DAPT, at early, but not later, stages of retinal development abolished both CD44-positive cells and Müller glial cells. Together, CD44 was identified as an early cell surface marker of the Müller glia lineage, and Notch signalling was involved in commitment of retinal progenitor cells to CD44 positive Müller glial precursor cells.

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C-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis

Shimizu T., Ishikawa T., Sugihara E., Kuninaka S., Miyamoto T., Mabuchi Y., Matsuzaki Y., Tsunoda T., Miya F., Morioka H., Nakayama R., Kobayashi E., Toyama Y., Kawai A., Ichikawa H., Hasegawa T., Okada S., Ito T., Ikeda Y., Suda T., Saya H.

Oncogene 29 ( 42 ) 5687 - 5699 2010.10

ISSN 0950-9232

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (/) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

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Cancer stem cells in malignant glioma - The mechanism of cancer initiation and the therapeutic development

Nakano Ichiro, Saya Hideyuki

Neurological Surgery 38 ( 10 ) 879 - 889 2010.10

ISSN 0301-2603

Malignant glioma is one of the most lethal diseases in adulthood. The median survival of patients with the Grade IV glioma, glioblastoma multiforme (GBM), is shorter than 15 months and the current first-line therapies for this devastating disease have only a palliative effect. The cancer stem cell hypothesis has recently attracted a great deal of attention, owing to the promise of a novel cellular target for the treatment of tumors including GBM. Recent studies have demonstrated that existence of cancer stem cells in brain tumors (BTSC) accounts, at least in part, for the intractability of malignant glioma. From the therapeutic standpoint, characterization of the mechanism for tumor initiation and maintenance of the "stem-like state" of BTSC is crucial. However, multiple heterogeneous subtypes of cancer stem cells have recently been identified from malignant glioma, making the idea of cancer stem cell complicated. In addition, in some cancer types (e.g. melanoma), a considerable proportion of tumor cells may possess the stem cell property, indicating cancer stem cells may not be a rare cell population in tumors, at least in some organs. Based on the extensive genetic and epigenetic characterization of tumor growth mechanisms, various molecularly-targeted therapies have already been applied for patients, demonstrating a varying degree of success in cancer treatment. A significant improvement in patient prognosis was achieved in several cancer types including leukemia and breast cancer. It is no doubt that continuous effort is required to bring hope for patients with malignant glioma. In this study, we summarize the recent findings and approaches in the cancer stem cell field, mainly focusing on malignant glioma stem cells, and also describe potential future directions in this area.

Decreased expression of neurofibromin contributes to epithelial-mesenchymal transition in neurofibromatosis type 1

Arima Yoshimi, Hayashi Hidemi, Kamata Kanako, Goto Takaaki M., Sasaki Mikako, Kuramochi Akira, Saya Hideyuki

Experimental Dermatology 19 ( 8 ) 2010.08

ISSN 0906-6705

Plexiform and/or dermal neurofibromas are nerve sheath tumors of the peripheral nervous system that are usually present in individuals with neurofibromatosis type 1 (NF1). Neurofibromas arise from Schwann cells with biallelic inactivation of NF1, the gene that encodes neurofibromin. This protein is responsible for regulation of the Ras-mediated pathway, which has been shown to play a crucial role in epithelial-to-mesenchymal transition (EMT). EMT is a biological process that occurs during embryogenesis and wound healing and is involved in pathological processes such as organ fibrosis and cancer metastasis. However, the relationship between neurofibromin and EMT has not been elucidated. We investigated whether the EMT-related signaling pathway was upregulated in NF1-associated neurofibromas and Schwann cells by assessing the expression levels of the EMT-related transcription factors Snail, Slug, Twist, ZEB1 and ZEB2. Immunohistochemical studies and quantitative reverse transcription polymerase chain reaction revealed an increase in the expression levels of EMT-related transcription factors in neurofibroma specimens and NF1-derived Schwann cells (sNF96.2). In addition, the silencing of NF1 by siRNA induced the expression of EMT-related transcription factors in normal human Schwann cells and in epithelial-like breast cancer cells. Our findings suggest that the loss of neurofibromin activated the EMT-related signaling pathway and that the excessive mesenchymal reaction may play a key role in the development of NF1-associated neurofibromas.

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The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation

Naoe Hideaki, Araki Kimi, Nagano Osamu, Kobayashi Yusuke, Ishizawa Jo, Chiyoda Tatsuyuki, Shimizu Takatsune, Yamamura Ken Ichi, Sasaki Yutaka, Saya Hideyuki, Kuninaka Shinji

Molecular and Cellular Biology 30 ( 16 ) 3994 - 4005 2010.08

ISSN 0270-7306

Cdh1 is an activator of the anaphase-promoting complex/cyclosome and contributes to mitotic exit and G₁ maintenance by targeting cell cycle proteins for degradation. However, Cdh1 is expressed and active in postmitotic or quiescent cells, suggesting that it has functions other than cell cycle control. Here, we found that homozygous Cdh1 gene-trapped (Cdh1 ^GT/GT) mouse embryonic fibroblasts (MEFs) and Cdh1-depleted HeLa cells reduced stress fiber formation significantly. The GTP-bound active Rho protein was apparently decreased in the Cdh1-depleted cells. The p190 protein, a major GTPase-activating protein for Rho, accumulated both in Cdh1 ^GT/GT MEFs and in Cdh1-knockdown HeLa cells. Cdh1 formed a physical complex with p190 and stimulated the efficient ubiquitination of p190, both in in vitro and in vivo. The motility of Cdh1-depleted HeLa cells was impaired; however, codepletion of p190 rescued the migration activity of these cells. Moreover, Cdh1^GT/GT embryos exhibited phenotypes similar to those observed for Rho-associated kinase I and II knockout mice: eyelid closure delay and disruptive architecture with frequent thrombus formation in the placental labyrinth layer, respectively. Furthermore, the p190 protein accumulated in the Cdh1^GT/GT embryonic tissues. Our data revealed a novel function for Cdh1 as a regulator of Rho and provided insights into the role of Cdh1 in cell cytoskeleton organization and cell motility.

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Proteome profiling reveals gender differences in the composition of human serum

Miike Koichiro, Aoki Masashi, Yamashita Ryo, Takegawa Yumiko, Saya Hideyuki, Miike Teruhisa, Yamamura Ken Ichi

Proteomics 10 ( 14 ) 2678 - 2691 2010.07

ISSN 1615-9853

Proteome analysis using human serum is a technological advancement that will enable the discovery of novel biomarkers and biomarker patterns of various human diseases. Although proteome analysis using serum has potential in disease prevention, early diagnosis and treatment of diseases, and evaluation of pharmacotherapies, this technology is still in its infancy. Thus, we sought to develop an advanced method of conducting proteome analysis on human serum. In this study, we report the development of the semi-comprehensive protein analytical technique, which involves the systematic use of iTRAQ labeling, HPLC, nano-LC and MS. We compared the composition of the serum proteome in males and females using this technique and detected gender-based differences in serum protein composition. This technology will enable the generation of databases that may ultimately lead to the discovery of specific biomarkers or biomarker patterns of various diseases.

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Breast cancer stem cells

Kai Kazuharu, Arima Yoshimi, Kamiya Toshio, Saya Hideyuki

Breast Cancer 17 ( 2 ) 80 - 85 2010.04

ISSN 1340-6868

Since the initial discovery of leukemia stem cells nearly a decade ago, a great deal of cancer research has focused on the identification of cancer stem cells (CSCs) in many types of solid tumors, including breast cancer. Through analysis of cell surface markers and xenotransplant models, a subpopulation of putative human breast cancer stem cells (BCSCs) that is CD24-negative/CD44- positive (CD24^-/ CD44⁺) and bears high aldehyde dehydrogenase 1 activity has been isolated in clinical samples of breast cancer tissues. Human BCSCs are considered to be derived from basal cells that reside in the basal membranes of alveolar units in human adult mammary glands. Furthermore, BCSCs have been shown to express higher levels of oxidative stress-responsive genes, which could confer part of their ability to resist anti-cancer therapy, than non-CSCs. The emerging picture of the biological properties of BCSCs would contribute for devising innovative therapies for breast cancer, targeting the intrinsic and extrinsic factors that maintain the BCSCs.

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Osteopontin-mediated enhanced hyaluronan binding induces multidrug resistance in mesothelioma cells

Tajima K., Ohashi R., Sekido Y., Hida T., Nara T., Hashimoto M., Iwakami S., Minakata K., Yae T., Takahashi F., Saya H., Takahashi K.

Oncogene 29 ( 13 ) 1941 - 1951 2010.04

ISSN 0950-9232

Malignant pleural mesothelioma (MPM) is resistant to chemotherapy and thus shows a dismal prognosis. Osteopontin (OPN), a secreted noncollagenous and phosphoprotein, is suggested to be involved in the pathogenesis of MPM. However, the precise role of OPN, especially in the multidrug resistance of MPM, remains to be elucidated. We therefore established stable transfectants (ACC-MESO-1/OPN), which constitutively express OPN, to determine its role in the chemoresistance observed in MPM. The introduction of the OPN gene provides MPM cells with upregulated multidrug resistance through the mechanism of enhanced hyaluronate (HA) binding. The expression of CD44 variant isoforms, which inhibit HA binding, significantly decreased in ACC-MESO-1/OPN cells in comparison to control transfectants. Interestingly, the inhibition of the HA-CD44 interaction abrogated multidrug resistance in the ACC-MESO-1/OPN, thus suggesting the involvement of the surviving signal emanating from the HA-CD44 interaction. An enhanced level of the p-Akt in ACC-MESO-1/OPN cells was observed, and was diminished by CD44 siRNA. Inhibition of the Akt phosphorylation increased in number of the cells underwent apoptosis induced by NVB, VP-16 and GEM. Collectively, these results indicate that OPN is strongly involved in multidrug resistance by enhancing the CD44 binding to HA.

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CD44⁺ slow-cycling tumor cell expansion is triggered by cooperative actions of Wnt and prostaglandin E₂ in gastric tumorigenesis

Ishimoto Takatsugu, Oshima Hiroko, Oshima Masanobu, Kai Kazuharu, Torii Ryota, Masuko Takashi, Baba Hideo, Saya Hideyuki, Nagano Osamu

Cancer Science 101 ( 3 ) 673 - 678 2010.03

ISSN 1347-9032

Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow-cycling and, thereby, insensitive to chemo- and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44⁺ cells and slow-cycling cells and the underlying mechanisms for the emergence of CD44⁺ CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo-columnar junction (SCJ) in normal mouse stomach contains CD44⁺ stem cell-like slow-cycling cells and that this characteristic CD44⁺ gland was expanded by prostaglandin E2 (PGE₂) and Wnt signaling in K19-Wnt1/C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19-Wnt1, K19-C2mE and K19-Wnt1/C2mE, revealed that the expansion of CD44⁺ SCJ cells is triggered by PGE₂-mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44⁺ gland in gastric tumor of K19-Wnt1/C2mE mouse contains a few BrdU label-retaining quiescent or slow-cycling cells, suggesting that the CD44⁺ SCJ cells in normal mouse are candidates for the cell-of-origin of gastric CSCs. These observations suggest that PGE₂-mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44⁺ slow-cycling stem-like cells in SCJ, leading to development of lethal gastric tumors in mice.

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Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway

Techasen Anchalee, Loilome Watcharin, Namwat Nisana, Takahashi Eri, Sugihara Eiji, Puapairoj Anucha, Miwa Masanao, Saya Hideyuki, Yongvanit Puangrat

Cancer Science 101 ( 3 ) 658 - 665 2010.03

ISSN 1347-9032

Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real-time-polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini-associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log-rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12-. 0-tetradecanoyl phorbol-13-acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line-depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non-stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.

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Experimental therapies

Imamura Chiyo K., Takebe Naoko, Nakamura Seigo, Saya Hideyuki, Ueno Naoto T.

Nature Reviews Clinical Oncology 7 ( 3 ) 127 - 128 2010.03

ISSN 1759-4774

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Tumor necrosis factor-α regulates transforming growth factor-β-dependent epithelial-mesenchymal transition by promoting hyaluronan-CD44-moesin interaction

Takahashi Eri, Nagano Osamu, Ishimoto Takatsugu, Yae Toshifumi, Suzuki Yoshimi, Shinoda Takeshi, Nakamura Satoshi, Niwa Shinichiro, Ikeda Shun, Koga Hisashi, Tanihara Hidenobu, Saya Hideyuki

Journal of Biological Chemistry 285 ( 6 ) 4060 - 4073 2010.02

ISSN 0021-9258

Aberrant epithelial-mesenchymal transition (EMT) is involved in development of fibrotic disorders and cancer invasion. Alterations of cell-extracellular matrix interaction also contribute to those pathological conditions. However, the functional interplay between EMT and cell-extracellular matrix interactions remains poorly understood.Wenow show that the inflammatory mediator tumor necrosis factor-α (TNF-α) induces the formation of fibrotic foci by cultured retinal pigment epithelial cells through activation of transforming growth factor-β (TGF-β) signaling in a manner dependent on hyaluronan-CD44-moesin interaction. TNF-α promoted CD44 expression and moesin phosphorylation by protein kinase C, leading to the pericellular interaction of hyaluronan and CD44. Formation of the hyaluronan-CD44-moesin complex resulted in both cell-cell dissociation and increased cellular motility through actin remodeling. Furthermore, this complex was found to be associated with TGF-β receptor II and clathrin at actin microdomains, leading to activation of TGF-β signaling. We established an in vivo model of TNF-α-induced fibrosis in the mouse eye, and such ocular fibrosis was attenuated in CD44-null mice. The production of hyaluronan and its interaction with CD44, thus, play an essential role in TNF-α-induced EMT and are potential therapeutic targets in fibrotic disorders.

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PSF1, a DNA replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties

Nagahama Yumi, Ueno Masaya, Miyamoto Satoru, Morii Eiichi, Minami Takashi, Mochizuki Naoki, Saya Hideyuki, Takakura Nobuyuki

Cancer Research 70 ( 3 ) 1215 - 1224 2010.02

ISSN 0008-5472

PSF1 (partner of sld five 1) is an evolutionarily conserved DNA replication factor implicated in DNA replication in lower species that is strongly expressed in a wide range of normal stem cell populations and progenitor cell populations. Because stem and progenitor cells possess high proliferative capacity, we hypothesized that PSF1 may play an important role in tumor growth. To begin to investigate PSF1 function in cancer cells, we cloned the mouse PSF1 promoter and generated lung and colon carcinoma cells that stably express a PSF1 promoter-reporter gene. Reporter expression in cells correlated with endogenous PSF1 mRNA expression. In a tumor cell xenograft model, high levels of reporter expression correlated with high proliferative activity, serial transplantation potential, and metastatic capability. Notably, cancer cells expressing reporter levels localized to perivascular regions in tumors and displayed expression signatures related to embryonic stem cells. RNAi-mediated silencing of endogenous PSF1 inhibited cancer cell growth by disrupting DNA synthesis and chromosomal segregation. These findings implicate PSF1 in tumorigenesis and offer initial evidence of its potential as a theranostic target.

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Molecularly targeted therapies for glioma

Yamanaka Ryuya, Saya Hideyuki

Annals of Neurology 66 ( 6 ) 717 - 729 2009.12

ISSN 0364-5134

Over the past decade, molecularly targeted therapies have been added to cytotoxic and antiendocrine drugs in the treatment of cancer, with the aim of targeting the molecular pathways that underlie the carcinogenic process and maintain the cancer phenotype. Success with some of these agents has suggested that identification and validation of drug targets is the starting point for the development of active, safe, and effective drugs. The main molecular targets used to develop anticancer drugs are cell surface receptors, signal transduction pathways, gene transcription targets, ubiquitin-proteasome/heat shock proteins, and tumor micro-environment components. Here, we review the development of the main molecularly targeted noncytotoxic agents studied in glioma, highlighting lessons derived from the development of these novel drugs and proposing new horizons for the clinical development of molecularly targeted therapies.

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Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model

Kai Kazuharu, Nagano Osamu, Sugihara Eiji, Arima Yoshimi, Sampetrean Oltea, Ishimoto Takatsugu, Nakanishi Masaya, Ueno Naoto T., Iwase Hirotaka, Saya Hideyuki

Cancer Science 100 ( 12 ) 2275 - 2282 2009.12

ISSN 1347-9032

The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self-renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non-CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133-positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating-sphere-derived cells, which are known to be the cells of enriching CSCs. Sphere-derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESA^lowCD44^-/low and ESA^highCD44^high) exhibited a biological/proliferative hierarchy of sphere-forming and soft agar colony-forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESA^lowCD44^-/low cells were allowed to aggregate and re-form floating-spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating-spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model.

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