Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Suppression of centrosome amplification after DNA damage depends on p27 accumulation

Sugihara Eiji, Kanai Masayuki, Saito Soichiro, Nitta Takayuki, Toyoshima Hideo, Nakayama Keiko, Nakayama Keiichi I., Fukasawa Kenji, Schwab Manfred, Saya Hideyuki, Miwa Masanao

Cancer Research 66 ( 8 ) 4020 - 4029 2006.04

ISSN 0008-5472

The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21^Cip1 and p27^Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after γ-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27^-/- and p27^+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability.

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Antitumor effect of E1A in ovarian cancer by cytoplasmic sequestration of activated ERK by PEA15

Bartholomeusz C., Itamochi H., Nitta M., Saya H., Ginsberg M. H., Ueno N. T.

Oncogene 25 ( 1 ) 79 - 90 2006.01

ISSN 0950-9232

The adenovirus type 5 gene E1A is known to suppress tumorigenicity by transcriptionally downregulating HER-2/neu (HER2) or by inducing apoptosis. We show here that E1A also suppressed the tumorigenicity of the low-HER2-expressing ovarian cancer cell line OVCAR-3 by decreasing cell proliferation. We further found that the mechanism responsible for this reduced proliferation is the presence of PEA15 (phosphoprotein enriched in astrocytes), which is upregulated by E1A in ovarian cancer; PEA15 promotes translocation of ERK from the nucleus to the cytoplasm, leading to inhibition of ERK-dependent transcription and proliferation. Indeed, siRNA-mediated knockdown of PEA15 expression in OVCAR-3 stable E1A transfectants resulted in a nuclear accumulation of the active form of ERK, followed by an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. Finally, PEA15 by itself suppressed colony formation in breast and ovarian cancer cell lines, in which E1A is known to have antitumor activity. We conclude that part of the antitumor effect of E1A in ovarian cancer results from cytoplasmic sequestration of the activated form of ERK by PEA15.

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Preliminary individualized chemotherapy for malignant astrocytomas based on O⁶-methylguanine-deoxyribonucleic acid methyltransferase methylation analysis

Saya Hideyuki

Neurologia Medico-Chirurgica 46 ( 8 ) 394 2006

ISSN 0470-8105

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The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway

Ozawa Tatsuya, Araki Norie, Yunoue Shunji, Tokuo Hiroshi, Feng Liping, Patrakitkomjorn Siriporn, Hara Toshihiro, Ichikawa Yasuko, Matsumoto Kunio, Fuji Kiyotaka, Saya Hideyuki

Journal of Biological Chemistry 280 ( 47 ) 39524 - 39533 2005.11

ISSN 0021-9258

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

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The tumor suppressor WARTS activates the Omi/HtrA2-dependent pathway of cell death

Kuninaka Shinji, Nomura Masanobu, Hirota Toru, Iida Shin Ichi, Hara Toshihiro, Honda Shinobu, Kunitoku Naoko, Sasayama Takashi, Arima Yoshimi, Marumoto Tomotoshi, Koja Kageharu, Yonehara Shin, Saya Hideyuki

Oncogene 24 ( 34 ) 5287 - 5298 2005.08

ISSN 0950-9232

Drosophila tumor suppressor WARTS (Wts) is an evolutionally conserved serine/threonine kinase and parti cipates in a signaling complex that regulates both proliferation and apoptosis to ensure the proper size and shape of the fly. Human counterparts of this complex have been found to be frequently downregulated or mutated in cancers. WARTS, a human homolog of Wts, is also known as tumor suppressor and mitotic regulator, but its molecular implications in tumorigenesis are still obscure. Here, we show that WARTS binds via its C-terminus to the PDZ domain of a proapoptotic serine protease Omi/ HtrA2. Depletion of WARTS inhibited Omi/HtrA2-mediated cell death, whereas overexpression of WARTS promoted this process. Furthermore, WARTS can enhance the protease activity of Omi/HtrA2 both in vivo and in vitro. Activation of Omi/HtrA2-mediated cell death is thus a potential mechanism for the tumor suppressive activity of WARTS.

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Multiple roles of vertebrate REV genes in DNA repair and recombination

Okada Takashi, Sonoda Eiichiro, Yoshimura Michio, Kawano Yoshiaki, Saya Hideyuki, Kohzaki Masaoki, Takeda Shunichi

Molecular and Cellular Biology 25 ( 14 ) 6103 - 6111 2005.07

ISSN 0270-7306

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1 ^-/- REV3^-/- REV7^-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1^-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polζ (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.

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Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Sasayama Takashi, Marumoto Tomotoshi, Kunitoku Naoko, Zhang Dongwei, Tamaki Norihiko, Kohmura Eiji, Saya Hideyuki, Hirota Toru

Genes to Cells 10 ( 7 ) 627 - 638 2005.07

ISSN 1356-9597

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB.We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were overexpressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.

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Molecular mechanism of tumorigenesis by cell cycle regulation and new therapeutical approaches

Saya Hideyuki

Japanese Journal of Neurosurgery 14 ( 6 ) 373 - 378 2005.06

ISSN 0917-950X

Cell cycle checkpoints prevent transition from one phase of the cell cycle to the next until all processes of the present phase are completed. Defects in the checkpoint functions result in gene mutations and chromosome damages, which contribute to the development and progression of tumors. However, loss of checkpoint function in some cancer cells is considered to be associated with their sensitivity to antineoplastic treatments such as chemotherapy and radiation therapy. Most cancer therapies target cell cycle checkpoints by activating checkpoint-mediated cell death or by enhancing chemical sensitivity due to loss of checkpoint function. By treatment with genotoxic agents, cancer cells, which generally have impairment of checkpoint functions, initially arrest in the G2 phase of the cell cycle but are unable to maintain cell-cycle arrest. Those cells eventually die as they entered mitosis. This process is called 'mitotic catastrophe'. This review discusses the critical relationship between mitotic checkpoint function and sensitivity of cancer cells to anti-tumor therapies.

Transcriptional blockade induces p53-dependent apoptosis associated with translocation of p53 to mitochondria

Arima Yoshimi, Nitta Masayuki, Kuninaka Shinji, Zhang Dongwei, Fujiwara Toshiyoshi, Taya Yoichi, Nakao Mitsuyoshi, Saya Hideyuki

Journal of Biological Chemistry 280 ( 19 ) 19166 - 19176 2005.05

ISSN 0021-9258

The tumor suppressor p53 functions as a transcriptional activator to induce cell cycle arrest and apoptosis in response to BNA damage. Although p53 was also shown to mediate apoptosis in a manner independent of its transactivation activity, the mechanism and conditions that trigger such cell death have remained largely unknown. We have now shown that inhibition of RNA polymerase II-mediated transcription by α-amanitin or RNA interference induced p53-dependent apoptosis. Inhibition of pol II-mediated transcription resulted in down-regulation of p21^Cip1, which was caused by both transcriptional suppression and protein degradation, despite eliciting p53 accumulation, allowing the cells to progress into S phase and then to undergo apoptosis. This cell death did not require the transcription of p53 target genes and was preceded by translocation of the accumulated p53 to mitochondria. Our data thus suggested that blockade of pol II-mediated transcription induced p53 accumulation in mitochondria and was the critical factor for eliciting p53-dependent but transcription-independent apoptosis.

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RNA interference targeting aurora kinase A suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells

Hata Tatsuo, Furukawa Toru, Sunamura Makoto, Egawa Shinichi, Motoi Fuyuhiko, Ohmura Noriyuki, Marumoto Tomotoshi, Saya Hideyuki, Horii Akira

Cancer Research 65 ( 7 ) 2899 - 2905 2005.04

ISSN 0008-5472

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G₂-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G₂-M transition, by the RNA interference-mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

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Chfr is required for tumor suppression and Aurora A regulation

Yu Xiaochun, Minter-Bykhouse Katherine, Malureanu Liviu, Zhao Wei Meng, Zhang Dongwei, Merkle Carolin J., Ward Irene M., Saya Hideyuki, Fang Guowei, Van Deursen Jan, Chen Junjie

Nature Genetics 37 ( 4 ) 401 - 406 2005.04

ISSN 1061-4036

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

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Aurora-A - A guardian of poles

Marumoto Tomotoshi, Zhang Dongwei, Saya Hideyuki

Nature Reviews Cancer 5 ( 1 ) 42 - 50 2005.01

ISSN 1474-175X

The three human homologues of Aurora kinases (A, B and C) are essential for proper execution of various mitotic events and are important for maintaining genomic integrity. Aurora-A is mainly localized at spindle poles and the mitotic spindle during mitosis, where it regulates the functions of centrosomes, spindles and kinetochores required for proper mitotic progression. Recent studies have revealed that Aurora-A is frequently overexpressed in various cancer cells, indicating its involvement in tumorigenesis. What are the normal physiological roles of Aurora-A, how are these regulated and how might the enzyme function during tumorigenesis?

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Molecular mechanisms of brain tumor invasion

Saya Hideyuki

Nippon rinsho. Japanese journal of clinical medicine 63 Suppl 9 61 - 67 2005

ISSN 0047-1852

Cell Death

Saya Hideyuki

Advances in Bioethics 8 209 - 223 2005

ISSN 1479-3709

Natural selection is the mechanism of evolution originally proposed by Charles Darwin. This process is driven by mutation and death. Therefore, "individual death" is a critical event for evolution of species. Analogous to the significance of individual death in species evolution, recent biological observations have revealed that "cell death" is an important event for maintenance of individuals and offspring. Cell death is caused not only by injuries and pathological conditions, but also by programed intracellular signals. This type of programed cell death is called "apoptosis." Apoptosis signals are regulated in mitochondria, eukaryotic organelles of symbiotic origin, and play a critical role in survival of individuals by eliminating some cells.

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Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein

Koga Hisashi, Izawa Ichiro, Araki Norie, Saya Hideyuki, Mimori Tatsuyuki

Microbiology and Immunology 49 ( 4 ) 297 - 302 2005

ISSN 0385-5600

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, ¹²⁵I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.

Overexpression of heparin-binding growth-associated molecule in malignant glioma cells

Ducati Alessandro, Mut Melike, Shaffrey Mark E., Laws Edward R., Saya Hideyuki

Neurologia Medico-Chirurgica 44 ( 12 ) 644 - 645 2004.12

ISSN 0470-8105

Mechanism and biological significance of CD44 cleavage

Nagano Osamu, Saya Hideyuki

Cancer Science 95 ( 12 ) 930 - 935 2004.12

ISSN 1347-9032

There are multiple steps in the metastasis of cancer cells. Tumor cells must first detach from the tumor mass and invade the surrounding extracellular matrix (ECM). In this step, cell surface adhesion molecules play an important role in the interaction between the cells and their microenvironments. CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and is implicated in a wide variety of physiological and pathological processes. Recently, proteolytic cleavages of CD44 have been emerging as key regulatory events for the CD44 dependent cell-matrix interaction and signaling pathway. CD44 undergoes sequential proteolytic cleavages in the ectodomain and intramembranous domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. The ectodomain cleavage of CD44 is triggered by multiple stimulations and contributes to the regulation of cell attachment to and migration on HA matrix. The ectodomain cleavage subsequently induces the intramembranous cleavage, which is mediated by presenilin (PS)-dependent γ-secretase. The intramembranous cleavage generates CD44ICD, which acts as a signal transduction molecule; it is translocated to the nucleus and activates transcription. An understanding of the underlying mechanism of these cleavages of CD44 could provide novel therapeutic targets for cancer cell invasion and metastasis.

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Cre-loxP-controlled periodic Aurora-A overexpression induces mitotic abnormalities and hyperplasia in mammary glands of mouse models

Zhang Dongwei, Hirota Toru, Marumoto Tomotoshi, Shimizu Michio, Kunitoku Naoko, Sasayama Takashi, Arima Yoshimi, Feng Liping, Suzuki Misao, Takeya Motohiro, Saya Hideyuki

Oncogene 23 ( 54 ) 8720 - 8730 2004.11

ISSN 0950-9232

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apopiosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.

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Mechanism and biological significance of sequential cleavages of CD44 - A potential molecular target for cancer therapy

Nagano Osamu, Murakami Daizo, Saya Hideyuki

Biotherapy 18 ( 5 ) 405 - 411 2004.09

ISSN 0914-2223

Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents

Nitta Masayuki, Kobayashi Osamu, Honda Shinobu, Hirota Toru, Kuninaka Shinji, Marumoto Tomotoshi, Ushio Yukitaka, Saya Hideyuki

Oncogene 23 ( 39 ) 6548 - 6558 2004.08

ISSN 0950-9232

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G₁ and G₂ checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint fonction by BnbR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

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