Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer

Arima Yoshimi, Hayashi Naoki, Hayashi Hidemi, Sasaki Mikako, Kai Kazuharu, Sugihara Eiji, Abe Eriko, Yoshida Atsushi, Mikami Shuji, Nakamura Seigo, Saya Hideyuki

International Journal of Cancer 130 ( 11 ) 2568 - 2579 2012.06

ISSN 0020-7136

Triple-negative breast cancer [TNBC, which is negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2] is a high-risk form of the disease without a specific therapy. DNA microarray and immunohistochemical analyses have shown that most TNBCs fall within the basal-like histological subset of breast cancers, which frequently exhibit inactivation of the retinoblastoma tumor suppressor (Rb) and upregulation of the cyclin-dependent kinase inhibitor p16 ^INK4a (p16). However, downregulation of p16 expression has been observed in some basal-like breast cancer cell lines, suggesting that such cells can be divided into two groups according to Rb and p16 status. We now show that cells that are CD44 ⁺ and CD24 ^-, a phenotype associated with stem-like breast cancer cells, are more abundant in ER ^-/p16 ^- breast cancer cell lines than in ER ^-/p16 ⁺ lines. It was also found that p16 expression was downregulated in mammospheres from an ER-negative breast cancer cell line. Depletion of p16 by RNA interference in ER-negative breast cancer cells increased the percentage of CD44 ⁺/CD24 ^- cells and increased the expression of mRNA of the ES-like genes Nanog, Oct4, and Sox2 through an Rb-independent pathway. Furthermore, such depletion of p16 reduced chemosensitivity. The loss of p16 expression may thus reduce the response of ER-negative breast cancer cells to chemotherapy by conferring cancer stem cell-like properties. Consistent with this conclusion, immunohistochemical analysis of the clinical samples suggests that low p16 expression in TNBC is associated with resistance to preoperative chemotherapy.

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LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

Chiyoda Tatsuyuki, Sugiyama Naoyuki, Shimizu Takatsune, Naoe Hideaki, Kobayashi Yusuke, Ishizawa Jo, Arima Yoshimi, Tsuda Hiroshi, Ito Masaaki, Kaibuchi Kozo, Aoki Daisuke, Ishihama Yasushi, Saya Hideyuki, Kuninaka Shinji

Journal of Cell Biology 197 ( 5 ) 625 - 641 2012.05

ISSN 0021-9525

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase- targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.

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Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion

Oikawa Tsukasa, Oyama Masaaki, Kozuka-Hata Hiroko, Uehara Shunsuke, Udagawa Nobuyuki, Saya Hideyuki, Matsuo Koichi

Journal of Cell Biology 197 ( 4 ) 553 - 568 2012.05

ISSN 0021-9525

Osteoclasts fuse to form multinucleated cells during osteoclastogenesis. This process is mediated by dynamic rearrangement of the plasma membrane and cytoskeleton, and it requires numerous factors, many of which have been identified. The underlying mechanism remains obscure, however. In this paper, we show that Tks5, a master regulator of invadopodia in cancer cells, is crucial for osteoclast fusion downstream of phosphoinositide 3-kinase and Src. Expression of Tks5 was induced during osteoclastogenesis, and prevention of this induction impaired both the formation of circumferential podosomes and osteoclast fusion without affecting cell differentiation. Tyrosine phosphorylation of Tks5 was attenuated in Src ^-/- osteoclasts, likely accounting for defects in podosome organization and multinucleation in these cells. Circumferential invadopodia formation in B16F0 melanoma cells was also accompanied by Tks5 phosphorylation. Co-culture of B16F0 cells with osteoclasts in an inflammatory milieu promoted the formation of melanoma-osteoclast hybrid cells. Our results thus reveal an unexpected link between circumferential podosome/invadopodium formation and cell-cell fusion in and beyond osteoclasts.

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Modulation of glucose metabolism by CD44 contributes to antioxidant status and drug resistance in cancer cells

Tamada Mayumi, Nagano Osamu, Tateyama Seiji, Ohmura Mitsuyo, Yae Toshifumi, Ishimoto Takatsugu, Sugihara Eiji, Onishi Nobuyuki, Yamamoto Takehiro, Yanagawa Hiroshi, Suematsu Makoto, Saya Hideyuki

Cancer Research 72 ( 6 ) 1438 - 1448 2012.03

ISSN 0008-5472

An increased glycolytic flux accompanied by activation of the pentose phosphate pathway (PPP) is implicated in chemoresistance of cancer cells. In this study, we found that CD44, a cell surface marker for cancer stem cells, interacts with pyruvate kinase M2 (PKM2) and thereby enhances the glycolytic phenotype of cancer cells that are either deficient in p53 or exposed to hypoxia. CD44 ablation by RNA interference increased metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis and the PPP. Such metabolic changes induced by CD44 ablation resulted in marked depletion of cellular reduced glutathione (GSH) and increased the intracellular level of reactive oxygen species in glycolytic cancer cells. Furthermore, CD44 ablation enhanced the effect of chemotherapeutic drugs in p53-deficient or hypoxic cancer cells. Taken together, our findings suggest that metabolic modulation by CD44 is a potential therapeutic target for glycolytic cancer cells that manifest drug resistance.

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RNA-binding protein musashi1 modulates glioma cell growth through the post-transcriptional regulation of notch and PI ₃ kinase/Akt signaling pathways

Muto Jun, Imai Takao, Ogawa Daisuke, Nishimoto Yoshinori, Okada Yohei, Mabuchi Yo, Kawase Takeshi, Iwanami Akio, Mischel Paul S., Saya Hideyuki, Yoshida Kazunari, Matsuzaki Yumi, Okano Hideyuki

PLoS One 7 ( 3 ) 2012.03

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P>0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI ₃ kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI ₃ kinase/Akt signaling.

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Induction of ZEB proteins by inactivation of RB protein is key determinant of mesenchymal phenotype of breast cancer

Arima Yoshimi, Hayashi Hidemi, Sasaki Mikako, Hosonaga Mari, Goto Takaaki M., Chiyoda Tatsuyuki, Kuninaka Shinji, Shibata Tatsuhiro, Ohata Hirokazu, Nakagama Hitoshi, Taya Yoichi, Saya Hideyuki

Journal of Biological Chemistry 287 ( 11 ) 7896 - 7906 2012.03

ISSN 0021-9258

Wepreviously showed that depletion of the retinoblastoma protein (RB) induces down-regulation of the adhesion molecule E-cadherin and thereby triggers the epithelial-mesenchymal transition. To further characterize the effect of RB inactivation on the phenotype of cancer cells, we have now examined RB expression in human breast cancer cell lines and clinical specimens. We found that RB-inactive cells exhibit a mesenchymal-like morphology and are highly invasive. We also found that ZEB proteins, transcriptional repressors of the E-cadherin gene, are markedly up-regulated in these cells in a manner sensitive to the miR-200 family of microRNAs. Moreover, depletion of ZEB in RB-inactive cells suppressed cell invasiveness and proliferation and induced epithelial marker expression. These results implicate ZEB in induction of the epithelial-mesenchymal transition, as well as in maintenance of the mesenchymal phenotype in RB-inactive cells. We also developed a screening program for inhibitors of ZEB1 expression and thereby identified several cyclin-dependent kinase inhibitors that blocked both ZEB1 expression and RB phosphorylation. Together, our findings suggest that RB inactivation contributes to tumor progression not only through loss of cell cycle control but also through up-regulation of ZEB expression and induction of an invasive phenotype.

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Fibroblast growth factor-2 is an important factor that maintains cellular immaturity and contributes to aggressiveness of osteosarcoma

Shimizu Takatsune, Ishikawa Tomoki, Iwai Sayaka, Ueki Arisa, Sugihara Eiji, Onishi Nobuyuki, Kuninaka Shinji, Miyamoto Takeshi, Toyama Yoshiaki, Ijiri Hiroshi, Mori Hajime, Matsuzaki Yumi, Yaguchi Tomonori, Nishio Hiroshi, Kawakami Yutaka, Ikeda Yasuo, Saya Hideyuki

Molecular Cancer Research 10 ( 3 ) 454 - 468 2012.03

ISSN 1541-7786

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal-regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma.

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Expression profiles of carcinosarcoma of the uterine corpus-are these similar to carcinoma or sarcoma?

Chiyoda Tatsuyuki, Tsuda Hiroshi, Tanaka Hideo, Kataoka Fumio, Nomura Hiroyuki, Nishimura Sadako, Takano Masashi, Susumu Nobuyuki, Saya Hideyuki, Aoki Daisuke

Genes Chromosomes and Cancer 51 ( 3 ) 229 - 239 2012.03

ISSN 1045-2257

Uterine carcinosarcoma (CS) is usually classified as uterine endometrial carcinoma (EC). However, CS is more aggressive even compared with high grade EC. CS is also reported to undergo epithelial to mesenchymal transition (EMT). In this study, we compared the gene expression profiles of CS, EC, and uterine sarcoma (US) and evaluated the role of EMT and chromosomal aberrations in CS tumor formation. Frozen tissues of 46 patients (14 CS, 24 EC, and 8 US) were included. The similarity was examined by Gene Set Enrichment Analysis (GSEA), Fisher's exact test, and clustering using "intrinsic gene set". We examined the expression of 39 EMT-related genes and evaluated TGF-beta signaling by phospho-SMAD2/3 (p-SMAD2/3) staining. Chromosomal regions differing between CS and EC were identified by chromosomal GSEA and comparative genomic hybridization (CGH) microarrays. Three statistical methods confirmed that CS resembled US rather than EC. Acquired markers of EMT were upregulated and attenuated markers of EMT were downregulated in CS. Immunohistochemistry showed that carcinomatous region of CS have higher expression of p-SMAD2/3 than EC (P = 0.008). Chromosomal GSEA showed that genes located at 19q13 had higher expression in CS. Furthermore, CGH microarray indicated that the TGFB1 locus at 19q13.1 was amplified in 4 of 7 samples. Based on the expression profile, CS resembles US rather than EC. TGF-beta signaling is activated in CS and chromosomal gains at 19q13, which includes the TGFB1 locus, suggest that this may contribute to high expression of TGF-beta and thereby EMT phenotype of CS.

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Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells

Masuko Kazue, Okazaki Shogo, Satoh Mayumi, Tanaka Goh, Ikeda Tatsuya, Torii Ryota, Ueda Eri, Nakano Takashi, Danbayashi Masaaki, Tsuruoka Tomoyo, Ohno Yoshiya, Yagi Hideki, Yabe Noritsugu, Yoshida Hideaki, Tahara Tomoyuki, Kataoka Shiro, Oshino Taichi, Shindo Takayuki, Niwa Shin ichiro, Ishimoto Takatsugu, Baba Hideo, Hashimoto Yoshiyuki, Saya Hideyuki, Masuko Takashi

PLoS One 7 ( 1 ) 2012.01

Background: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. Methods and Principal Findings: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. Conclusions: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.

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DNA damage signaling triggers degradation of histone methyltransferases through APC/C ^Cdh1 in senescent cells

Takahashi Akiko, Imai Yoshinori, Yamakoshi Kimi, Kuninaka Shinji, Ohtani Naoko, Yoshimoto Shin, Hori Satoshi, Tachibana Makoto, Anderton Emma, Takeuchi Takashi, Shinkai Yoichi, Peters Gordon, Saya Hideyuki, Hara Eiji

Molecular Cell 45 ( 1 ) 123 - 131 2012.01

ISSN 1097-2765

Both the DNA damage response (DDR) and epigenetic mechanisms play key roles in the implementation of senescent phenotypes, but very little is known about how these two mechanisms are integrated to establish senescence-associated gene expression. Here we show that, in senescent cells, the DDR induces proteasomal degradation of G9a and GLP, major histone H3K9 mono- and dimethyltransferases, through Cdc14B- and p21 ^Waf1/Cip1-dependent activation of APC/C ^Cdh1 ubiquitin ligase, thereby causing a global decrease in H3K9 dimethylation, an epigenetic mark for euchromatic gene silencing. Interestingly, induction of IL-6 and IL-8, major players of the senescence-associated secretory phenotype (SASP), correlated with a decline of H3K9 dimethylation around the respective gene promoters and knockdown of Cdh1 abolished IL-6/IL-8 expression in senescent cells, suggesting that the APC/C ^Cdh1-G9a/GLP axis plays crucial roles in aspects of senescent phenotype. These findings establish a role for APC/C ^Cdh1 and reveal how the DDR integrates with epigenetic processes to induce senescence-associated gene expression.

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In vivo assessment of cancerous tumors using boron doped diamond microelectrode

Fierro Stéphane, Yoshikawa Momoko, Nagano Osamu, Yoshimi Kenji, Saya Hideyuki, Einaga Yasuaki

Scientific Reports 2 2012

The in vitro and in vivo electrochemical detection of the reduced form of glutathione (L-γ-glutamyl-L-cysteinyl-glycine, GSH) using boron doped diamond (BDD) microelectrode for potential application in the assessment of cancerous tumors is presented. Accurate calibration curve for the determination of GSH could be obtained by the in vitro electrochemical measurements. Additionally, it was shown that it was possible to separate the detection of GSH from the oxidized form of glutathione (GSSG) using chronoamperometry measurements. In vivo GSH detection measurements have been performed in human cancer cells inoculated in immunodeficient mice. These measurements have shown that the difference of GSH level between cancerous and normal tissues can be detected. Moreover, GSH detection measurements carried out before and after X-ray irradiation have proved that it is possible to assess in vivo the decrease in GSH concentration in the tumor after a specific treatment.

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Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell

Yae Toshifumi, Tsuchihashi Kenji, Ishimoto Takatsugu, Motohara Takeshi, Yoshikawa Momoko, Yoshida Go J., Wada Takeyuki, Masuko Takashi, Mogushi Kaoru, Tanaka Hiroshi, Osawa Tsuyoshi, Kanki Yasuharu, Minami Takashi, Aburatani Hiroyuki, Ohmura Mitsuyo, Kubo Akiko, Suematsu Makoto, Takahashi Kazuhisa, Saya Hideyuki, Nagano Osamu

Nature Communications 3 2012

In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v⁺) subpopulation of 4T1 breast cancer cells, but not that of a CD44v^- subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v ⁺ cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.

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Molecular biology of glioma

Marumoto Tomotoshi, Saya Hideyuki

2 - 11 2012

Glioblastoma (GBM) is the most aggressive form of glioma. Despite ceaseless efforts by researchers and physicians to find new therapeutic strategies, there have been no significant advances in the treatment of GBMs for several decades and most patients with GBM die within one and half years of diagnosis. Undoubtedly, one reason for this is the insufficient understanding of the initiation and progression of GBMs at the molecular level. However, recent information regarding the genetic and epigenetic alterations and the microRNAs that are aberrantly activated or inactivated in GBMs has helped elucidate the formation of GBM in more detail. Here, we describe recent advances in the understanding of the biology of GBMs.

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Erratum

Tomita M., Toyota M., Ishikawa C., Nakazato T., Okudaira T., Matsuda T., Uchihara J. N., Taira N., Ohshiro K., Senba M., Tanaka Y., Ohshima K., Saya H., Tokino T., Mori N.

International Journal of Cancer 129 ( 11 ) 2763 2011.12

ISSN 0020-7136

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Transient depletion of p53 followed by transduction of c-Myc and K-Ras converts ovarian stem-like cells into tumor-initiating cells

Motohara Takeshi, Masuko Sachiko, Ishimoto Takatsugu, Yae Toshifumi, Onishi Nobuyuki, Muraguchi Teruyuki, Hirao Atsushi, Matsuzaki Yumi, Tashiro Hironori, Katabuchi Hidetaka, Saya Hideyuki, Nagano Osamu

Carcinogenesis 32 ( 11 ) 1597 - 1606 2011.11

ISSN 0143-3334

Although the existence of tumor-initiating cells (T-ICs) in several types of human cancer has been documented, the contribution of somatic stem cells to the development of T-ICs has remained unclear. Here, we show that normal mouse ovary contains epithelial cell adhesion molecule (EpCAM)-expressing stem-like cells that possess the ability to differentiate into cytokeratin 8 (CK8)-expressing epithelial progeny cells. Furthermore, RNA interference-mediated transient depletion of the tumor suppressor p53 followed by retrovirus-mediated transfer of c-Myc and K-Ras oncogenes in EpCAM-expressing ovarian stem-like cells resulted in the generation of ovarian T-ICs. The established ovarian T-ICs gave rise to hierarchically organized lethal tumors in vivo and were able to undergo peritoneal metastasis. Finally, subsequent RNA interference-mediated knockdown of p53 in tumor cells triggered the expansion of EpCAM-expressing stem-like tumor cells and induced further tumor growth. These data reveal a role for p53 in the development and expansion of ovarian stem-like tumor cells and subsequent malignant progression.

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Invasion precedes tumor mass formation in a malignant brain tumor model of genetically modified neural stem cells

Sampetrean Oltea, Saga Isako, Nakanishi Masaya, Sugihara Eiji, Fukaya Raita, Onishi Nobuyuki, Osuka Satoru, Akahata Masaki, Kai Kazuharu, Sugimoto Hachiro, Hirao Atsushi, Saya Hideyuki

Neoplasia 13 ( 9 ) 784 - 791 2011.09

ISSN 1522-8002

Invasiveness, cellular atypia, and proliferation are hallmarks of malignant gliomas. To effectively target each of these characteristics, it is important to understand their sequence during tumorigenesis. However, because most gliomas are diagnosed at an advanced stage, the chronology of gliomagenesis milestones is not well understood. The aim of the present study was to determine the onset of these characteristics during tumor development. Brain tumor-initiating cells (BTICs) were established by overexpressing H-Ras ^V12 in normal neural stem/progenitor cells isolated from the subventricular zone of adult mice harboring a homozygous deletion of the Ink4a/Arf locus. High-grade malignant brain tumors were then created by orthotopic implantation of 10 ⁵ BTICs into the forebrain of 6-week-old wild-type mice. Micewere killed every week for 5 weeks, and tumors were assessed for cellular atypia, proliferation, hemorrhage, necrosis, and invasion. All mice developed highly invasive, hypervascular glioblastoma-like tumors. A 100% penetrance rate and a 4-week median survival were achieved. Tumor cell migration along fiber tracts started within days after implantation and was followed by perivascular infiltration of tumor cells with marked recruitment of reactive host cells. Next, cellular atypia became prominent. Finally, mass proliferation and necrosis were observed in the last stage of the disease. Video monitoring of BTICs in live brain slices confirmed the early onset of migration, as well as the main cell migration patterns. Our results showed that perivascular and intraparenchymal tumor cell migration precede tumor mass formation in the adult brain, suggesting the need for an early and sustained anti-invasion therapy.

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Establishment of a choriocarcinoma model from immortalized normal extravillous trophoblast cells transduced with HRASV12

Kobayashi Yusuke, Shimizu Takatsune, Naoe Hideaki, Ueki Arisa, Ishizawa Joe, Chiyoda Tatsuyuki, Onishi Nobuyuki, Sugihara Eiji, Nagano Osamu, Banno Kouji, Kuninaka Shinji, Aoki Daisuke, Saya Hideyuki

American Journal of Pathology 179 ( 3 ) 1471 - 1482 2011.09

ISSN 0002-9440

Gestational choriocarcinoma is a malignant trophoblastic tumor. The development of novel molecular-targeted therapies is needed to reduce the toxicity of current multiagent chemotherapy and to treat successfully the chemoresistant cases. The molecular mechanisms underlying choriocarcinoma tumorigenesis remain uncharacterized, however, and appropriate choriocarcinoma animal models have not yet been developed. In this study, we established a choriocarcinoma model by inoculating mice with induced-choriocarcinoma cell1 (iC ³-1) cells, generated from HTR8/SVneo human trophoblastic cells retrovirally transduced with activated H-RAS (HRASV12). The iC ³-1 cells exhibited constitutive activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and developed into lethal tumors in all inoculated mice. Histopathological analysis revealed that the tumors consisted of two distinct types of cells, reminiscent of syncytiotrophoblasts and cytotrophoblasts, as seen in the human choriocarcinoma. The tumors expressed HLA-G and cytokeratin (trophoblast markers) and hCG (a choriocarcinoma marker). Comparative analysis of gene expression profiles between iC ³-1 cells and parental HTR8/SVneo cells revealed that iC ³-1 cells expressed matrix metalloproteinases, epithelialmesenchymal transition-related genes, and SOX3 at higher levels than parental trophoblastic cells. Administration of SOX3-specific short-hairpin RNA decreased SOX3 expression and attenuated the tumorigenic activity of iC ³-1 cells, suggesting that SOX3 overexpression might be critically involved in the pathogenesis of choriocarcinoma. Our murine model represents a potent new tool for studying the pathogenesis and treatment of choriocarcinoma.

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PRKAR1A is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma

Loilome Watcharin, Juntana Sirinun, Namwat Nisana, Bhudhisawasdi Vajarabhongsa, Puapairoj Anucha, Sripa Banchob, Miwa Masanao, Saya Hideyuki, Riggins Gregory J., Yongvanit Puangrat

International Journal of Cancer 129 ( 1 ) 34 - 44 2011.07

ISSN 0020-7136

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/β-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy.

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Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

Tabuse Masanao, Ohta Shigeki, Ohashi Yohei, Fukaya Raita, Misawa Aya, Yoshida Kazunari, Kawase Takeshi, Saya Hideyuki, Thirant Cécile, Chneiweiss Hérve, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Molecular Cancer 10 2011.05

Background: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.Results: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).Conclusions: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

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The cell cycle regulator Cdh1 controls the pool sizes of hematopoietic stem cells and mature lineage progenitors by protecting from genotoxic stress

Ishizawa Jo, Kuninaka Shinji, Sugihara Eiji, Naoe Hideaki, Kobayashi Yusuke, Chiyoda Tatsuyuki, Ueki Arisa, Araki Kimi, Yamamura Ken Ichi, Matsuzaki Yumi, Nakajima Hideaki, Ikeda Yasuo, Okamoto Shinichiro, Saya Hideyuki

Cancer Science 102 ( 5 ) 967 - 974 2011.05

ISSN 1347-9032

Various key cell cycle components, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators that maintain anaphase-promoting complex/cyclosome activity, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Here, we generated Cdh1 conditional gene-trap mice to examine Cdh1 functions in adult tissues by overcoming the embryonic lethality of Cdh1 homozygous gene-trap mice. We focused on the hematopoietic system and found that Cdh1-deficient mice exhibited a general decrease in mature lineage progenitor cells and a significant increase in short-term hematopoietic stem cells. This phenomenon became conspicuous by irradiation shortly after Cdh1 downregulation, suggesting that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells by protecting cells from genotoxic stress. We also found that the irradiation-induced G2/M checkpoint was defective in Cdh1-deficient BM cells, causing the loss of stem/progenitor cells. This is the first report revealing Cdh1 function in adult hematopoiesis and showing a role of Cdh1 in a G2/M checkpoint regulation in vivo.

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