Serendipity Workshop 2022, Best Poster Presentation Awards　　　　　　　　　　　　　　　
Identifying a novel therapeutic candidate for pancreatic cancer using a Drosophila model
Takako Ooshio

Concurrent targeting of GSK3 and MEK as a therapeutic strategy to treat pancreatic ductal adenocarcinoma
Junki Fukuda, Shinya Kosuge, Yusuke Satoh, Sho Sekiya, Ryodai Yamamura, Takako Ooshio, Taiga Hirata, Reo Sato, Kanako C. Hatanaka, Tomoko Mitsuhashi, Toru Nakamura, Yoshihiro Matsuno, Yutaka Hatanaka, Satoshi Hirano, Masahiro Sonoshita
Cancer Science, Wiley, 2024年02月06日
研究論文（学術雑誌）, Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide. However, drug discovery for PDAC treatment has proven complicated, leading to stagnant therapeutic outcomes. Here, we identify Glycogen synthase kinase 3 (GSK3) as a therapeutic target through a whole‐body genetic screening utilizing a ‘4‐hit’ Drosophila model mimicking the PDAC genotype. Reducing the gene dosage of GSK3 in a whole‐body manner or knocking down GSK3 specifically in transformed cells suppressed 4‐hit fly lethality, similar to Mitogen‐activated protein kinase kinase (MEK), the therapeutic target in PDAC we have recently reported. Consistently, a combination of the GSK3 inhibitor CHIR99021 and the MEK inhibitor trametinib suppressed the phosphorylation of Polo‐like kinase 1 (PLK1) as well as the growth of orthotopic human PDAC xenografts in mice. Additionally, reducing PLK1 genetically in 4‐hit flies rescued their lethality. Our results reveal a therapeutic vulnerability in PDAC that offers a treatment opportunity for patients by inhibiting multiple targets.

Drosophila Screening Identifies Dual Inhibition of MEK and AURKB as an Effective Therapy for Pancreatic Ductal Adenocarcinoma
Sho Sekiya, Junki Fukuda, Ryodai Yamamura, Takako Ooshio, Yusuke Satoh, Shinya Kosuge, Reo Sato, Kanako C. Hatanaka, Yutaka Hatanaka, Tomoko Mitsuhashi, Toru Nakamura, Yoshihiro Matsuno, Satoshi Hirano, Masahiro Sonoshita
Cancer Research, OF1, OF12, American Association for Cancer Research (AACR), 2023年06月28日
研究論文（学術雑誌）, Abstract

Significant progress has been made in understanding the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) by generating and using murine models. To accelerate drug discovery by identifying novel therapeutic targets on a systemic level, here we generated a Drosophila model mimicking the genetic signature in PDAC (KRAS, TP53, CDKN2A, and SMAD4 alterations), which is associated with the worst prognosis in patients. The ‘4-hit’ flies displayed epithelial transformation and decreased survival. Comprehensive genetic screening of their entire kinome revealed kinases including MEK and AURKB as therapeutic targets. Consistently, a combination of the MEK inhibitor trametinib and the AURKB inhibitor BI-831266 suppressed the growth of human PDAC xenografts in mice. In patients with PDAC, the activity of AURKB was associated with poor prognosis. This fly-based platform provides an efficient whole-body approach that complements current methods for identifying therapeutic targets in PDAC.

Significance:

Development of a Drosophila model mimicking genetic alterations in human pancreatic ductal adenocarcinoma provides a tool for genetic screening that identifies MEK and AURKB inhibition as a potential treatment strategy.

Hepatocyte-specific damage in acute toxicity of sodium ferrous citrate: Presentation of a human autopsy case and experimental results in mice　　　　　　　　　　　　　　　
Nishikawa Y, Matsuo Y, Watanabe R, Miyazato M, Matsuo M, Nagahama Y, Tanaka H, Ooshio T, Goto M, Okada Y, Fujita S
Toxocol Rep, 10, 669, 679, 2023年05月, ［査読有り］
英語

High levels of Myc expression are required for the robust proliferation of hepatocytes, but not for the sustained weak proliferation.
Masanori Goto, Takako Ooshio, Masahiro Yamamoto, Hiroki Tanaka, Yumiko Fujii, Lingtong Meng, Yuki Kamikokura, Yoko Okada, Yuji Nishikawa
Biochimica et biophysica acta. Molecular basis of disease, 1869, 4, 166644, 166644, 2023年01月18日, ［国際誌］
英語, 研究論文（学術雑誌）, In contrast to the robust proliferation exhibited following acute liver injury, hepatocytes exhibit long-lasting proliferative activity in chronic liver injury. The mechanistic differences between these distinct modes of proliferation are unclear. Hepatocytes exhibited robust proliferation that peaked at 2 days following partial hepatectomy in mice, but this proliferation was completely inhibited by hepatocyte-specific expression of MadMyc, a Myc-suppressing chimeric protein. However, Myc suppression induced weak but continuous hepatocyte proliferation, thereby resulting in full restoration of liver mass despite an initial delay. Late-occurring proliferation was accompanied by prolonged suppression of proline dehydrogenase (PRODH) expression, and forced PRODH overexpression inhibited hepatocyte proliferation. In hepatocytes in chronic liver injury, Myc was not activated but PRODH expression was suppressed in regenerating hepatocytes. In liver tumors, PRODH expression was often suppressed, especially in the highly proliferative tumors with distinct Myc expression. Our results indicate that the robust proliferation of hepatocytes following acute liver injury requires high levels Myc expression and that there is a compensatory Myc-independent mode of hepatocyte proliferation with the regulation of proline metabolism, which might be relevant to liver regeneration in chronic injury.

Generation of combined hepatocellular-cholangiocarcinoma through transdifferentiation and dedifferentiation in p53-knockout mice.
Yang Liu, Bing Xin, Masahiro Yamamoto, Masanori Goto, Takako Ooshio, Yuki Kamikokura, Hiroki Tanaka, Lingtong Meng, Yoko Okada, Yusuke Mizukami, Yuji Nishikawa
Cancer science, 112, 8, 3111, 3124, 2021年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The two principal histological types of primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma, can coexist within a tumor, comprising combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Although the possible involvement of liver stem/progenitor cells has been proposed for the pathogenesis of cHCC-CCA, the cells might originate from transformed hepatocytes that undergo ductular transdifferentiation or dedifferentiation. We previously demonstrated that concomitant introduction of mutant HRASV12 (HRAS) and Myc into mouse hepatocytes induced dedifferentiated tumors that expressed fetal/neonatal liver genes and proteins. Here, we examine whether the phenotype of HRAS- or HRAS/Myc-induced tumors might be affected by the disruption of the Trp53 gene, which has been shown to induce biliary differentiation in mouse liver tumors. Hepatocyte-derived liver tumors were induced in heterozygous and homozygous p53-knockout (KO) mice by hydrodynamic tail vein injection of HRAS- or Myc-containing transposon cassette plasmids, which were modified by deleting loxP sites, with a transposase-expressing plasmid. The HRAS-induced and HRAS/Myc-induced tumors in the wild-type mice demonstrated histological features of HCC, whereas the phenotype of the tumors generated in the p53-KO mice was consistent with cHCC-CCA. The expression of fetal/neonatal liver proteins, including delta-like 1, was detected in the HRAS/Myc-induced but not in the HRAS-induced cHCC-CCA tissues. The dedifferentiation in the HRAS/Myc-induced tumors was more marked in the homozygous p53-KO mice than in the heterozygous p53-KO mice and was associated with activation of Myc and YAP and suppression of ERK phosphorylation. Our results suggest that the loss of p53 promotes ductular differentiation of hepatocyte-derived tumor cells through either transdifferentiation or Myc-mediated dedifferentiation.

Tiny Drosophila makes giant strides in cancer research.
Ryodai Yamamura, Takako Ooshio, Masahiro Sonoshita
Cancer science, 112, 2, 505, 514, 2021年02月, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Cancer burden has been increasing worldwide, making cancer the second leading cause of death in the world. Over the past decades, various experimental models have provided important insights into the nature of cancer. Among them, the fruit fly Drosophila as a whole-animal toolkit has made a decisive contribution to our understanding of fundamental mechanisms of cancer development including loss of cell polarity. In recent years, scalable Drosophila platforms have proven useful also in developing anti-cancer regimens that are effective not only in mammalian models but also in patients. Here, we review studies using Drosophila as a tool to advance cancer study by complementing other traditional research systems.

Essential role of autophagy in protecting neonatal haematopoietic stem cells from oxidative stress in a p62-independent manner.
Naho Nomura, Chiaki Ito, Takako Ooshio, Yuko Tadokoro, Susumu Kohno, Masaya Ueno, Masahiko Kobayashi, Atsuko Kasahara, Yusuke Takase, Kenta Kurayoshi, Sha Si, Chiaki Takahashi, Masaaki Komatsu, Toru Yanagawa, Atsushi Hirao
Scientific reports, 11, 1, 1666, 1666, 2021年01月18日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Autophagy is a cellular degradation system contributing to homeostasis of tissue stem cells including haematopoietic stem cells (HSCs). It plays pleiotropic roles in HSC characteristics throughout life, but its stage-specific roles in HSC self-renewal are unclear. To investigate the effects of Atg5 deletion on stage-specific HSC functions, we compared the repopulating capacity of HSCs in Atg5f/f;Vavi-cre mice from postnatal day (P) 0-7 weeks of age. Interestingly, Atg5 deficiency led to no remarkable abnormality in the HSC self-renewal capacity at P0, but significant defects at P7, followed by severe defects. Induction of Atg5 deletion at P5 by tamoxifen administration to Atg5f/f;Rosa26-Cre-ERT2 mice resulted in normal haematopoiesis, including the HSC population, until around 1 year, suggesting that Atg5 in the early neonatal period was critical for haematopoiesis in adults. Mitochondrial oxidative stress was increased by Atg5 loss in neonatal HSC/progenitor cells. Although p62 had accumulated in immature bone marrow cells of Atg5f/f;Vavi-cre mice, p62 deletion did not restore defective HSC functions, indicating that Atg5-dependent haematopoietic regulation in the developmental period was independent of p62. This study proposes a critical role of autophagy in HSC protection against harsh environments in the early neonatal stage, which is essential for healthy long-term haematopoiesis.

p53ノックアウトマウス肝細胞のin vitro形質転換 肝内移植による胆管分化を伴う腫瘍の形成　　　　　　　　　　　　　　　
上小倉 佑機, 後藤 正憲, 人見 淳一, 藤井 裕美子, 田中 宏樹, 渡辺 賢二, 大塩 貴子, 山本 雅大, 孟 玲童, 岡田 陽子, 西川 祐司
日本癌学会総会記事, 79回, PJ2, 2, (一社)日本癌学会, 2020年10月
英語

Hepatocyte MKK7 Contributes to Restoration of the Liver Parenchyma Following Injury
Takako Ooshio, Masahiro Yamamoto, Kiyonaga Fujii, Bing Xin, Kenji Watanabe, Masanori Goto, Yoko Okada, Akira Suzuki, Josef M. Penninger, Hiroshi Nishina, Yuji Nishikawa
Hepatology, 73, 6, 2510, 2526, Wiley, 2020年09月23日, ［査読有り］, ［筆頭著者］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND AND AIMS: Mitogen-activated protein kinase kinase (MKK) 7 and MKK4 are upstream activators of c-Jun NH2 -terminal kinases (JNKs) and have been shown to be required for the early development of the liver. Although it has been suggested that MKK7 might be involved in the regulation of hepatocyte proliferation, the functional role of MKK7 in the liver has remained unclear. APPROACH AND RESULTS: Here, we examined phenotypic alterations in liver-specific or hepatocyte/hematopoietic cell-specific MKK7 knockout (KO) mice, which were generated by crossing MKK7LoxP/LoxP with albumin-cyclization recombination (Alb-Cre) or myxovirus resistance protein 1-Cre mice, respectively. The livers of Alb-Cre-/+ MKK7LoxP/LoxP mice developed without discernible tissue disorganization. MKK7 KO mice responded normally to liver injuries incurred by partial hepatectomy or injection of CCl4 . However, tissue repair following CCl4 -induced injury was delayed in MKK7 KO mice compared with that of control mice. Furthermore, after repeated injections of CCl4 for 8 weeks, the liver in MKK7 KO mice showed intense fibrosis with increased protractive hepatocyte proliferation, suggesting that MKK7 deficiency might affect regenerative responses of hepatocytes in the altered tissue microenvironment. MKK7 KO hepatocytes demonstrated normal proliferative activity when cultured in monolayers. However, MKK7 KO significantly suppressed branching morphogenesis of hepatocyte aggregates within a collagen gel matrix. Microarray analyses revealed that suppression of branching morphogenesis in MKK7 KO hepatocytes was associated with a reduction in mRNA expression of transgelin, glioma pathogenesis related 2, and plasminogen activator urokinase-type (Plau); and forced expression of these genes in MKK7 KO hepatocytes partially recovered the attenuated morphogenesis. Furthermore, hepatocyte-specific overexpression of Plau rescued the impaired tissue repair of MKK7 KO mice following CCl4 -induced injury. CONCLUSIONS: MKK7 is dispensable for the regenerative proliferation of hepatocytes but plays important roles in repair processes following parenchymal destruction, possibly through modulation of hepatocyte-extracellular matrix interactions.

Emergence of the Dedifferentiated Phenotype in Hepatocyte-Derived Tumors in Mice: Roles of Oncogene-Induced Epigenetic Alterations.
Watanabe K, Yamamoto M, Xin B, Ooshio T, Goto M, Fujii K, Liu Y, Okada Y, Furukawa H, Nishikawa Y
Hepatology communications, 3, 5, 697, 715, 2019年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Hepatocellular carcinoma often reactivates the genes that are transiently expressed in fetal or neonatal livers. However, the mechanism of their activation has not been elucidated. To explore how oncogenic signaling pathways could be involved in the process, we examined the expression of fetal/neonatal genes in liver tumors induced by the introduction of myristoylated v-akt murine thymoma viral oncogene (AKT), HRas proto-oncogene, guanosine triphosphatase (HRASV12), and MYC proto-oncogene, bHLH transcription factor (Myc), in various combinations, into mouse hepatocytes in vivo. Distinct sets of fetal/neonatal genes were activated in HRAS- and HRAS/Myc-induced tumors: aldo-keto reductase family 1, member C18 (Akr1c18), glypican 3 (Gpc3), carboxypeptidase E (Cpe), adenosine triphosphate-binding cassette, subfamily D, member 2 (Abcd2), and trefoil factor 3 (Tff3) in the former; insulin-like growth factor 2 messenger RNA binding protein 3 (Igf2bp3), alpha fetoprotein (Afp), Igf2, and H19, imprinted maternally expressed transcript (H19) in the latter. Interestingly, HRAS/Myc-induced tumors comprised small cells with a high nuclear/cytoplasmic ratio and messenger RNA (mRNA) expression of delta-like noncanonical Notch ligand 1 (Dlk1), Nanog homeobox (Nanog), and sex determining region Y-box 2 (Sox2). Both HRAS- and HRAS/Myc-induced tumors showed decreased DNA methylation levels of Line1 and Igf2 differentially methylated region 1 and increased nuclear accumulation of 5-hydroxymethylcytosine, suggesting a state of global DNA hypomethylation. HRAS/Myc-induced tumors were characterized by an increase in the mRNA expression of enzymes involved in DNA methylation (DNA methyltransferase [Dnmt1, Dnmt3]) and demethylation (ten-eleven-translocation methylcytosine dioxygenase 1 [Tet1]), sharing similarities with the fetal liver. Although mouse hepatocytes could be transformed by the introduction of HRAS/Myc in vitro, they did not express fetal/neonatal genes and sustained global DNA methylation, suggesting that the epigenetic alterations were influenced by the in vivo microenvironment. Immunohistochemical analyses demonstrated that human hepatocellular carcinoma cases with nuclear MYC expression were more frequently positive for AFP, IGF2, and DLK1 compared with MYC-negative tumors. Conclusion: The HRAS signaling pathway and its interactions with the Myc pathway appear to reactivate fetal/neonatal gene expression in hepatocytic tumors partly through epigenetic alterations, which are dependent on the tumor microenvironment.

Oncogenic Determination of a Broad Spectrum of Phenotypes of Hepatocyte-Derived Mouse Liver Tumors
Masahiro Yamamoto, Bing Xin, Kenji Watanabe, Takako Ooshio, Kiyonaga Fujii, Xi Chen, Yoko Okada, Hiroaki Abe, Yoshimitsu Taguchi, Naoyuki Miyokawa, Hiroyuki Furukawa, Yuji Nishikawa
AMERICAN JOURNAL OF PATHOLOGY, 187, 12, 2711, 2725, ELSEVIER SCIENCE INC, 2017年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Activation of the phosphoinositide 3-kinase- AKT, Yes-associated protein (YAP), and MYC pathways is involved in human liver cancers, including hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). However, the nature of the interactions among these pathways has remained poorly understood. Herein, we demonstrate the coordination of these pathways during the formation of mouse liver tumors induced by hepatocyte-specific somatic integration of myristoylated AKT, mutant YAP, Myc, or their combinations. Although the introduction of YAP or Myc alone was inefficient in inducing tumors, these proteins accelerated tumorigenesis induced by AKT. The generated tumors demonstrated various histological features: low-grade HCC by AKT/Myc, CC by AKT/YAP, and high-grade HCC by AKT/Myc/YAP. CC induced by AKT/YAP was associated with activation of the Notch pathway. Interestingly, the combination of Myc and YAP generated tumors composed of hepatoblast/stem-like cells expressing mRNA for Afp, D1k1, Nanog, and Sox2 and occasionally forming immature ducts. Finally, immunohistochemical analysis revealed that human HCC and CC were predominantly associated with phosphorylation of S6 and glycogen synthase kinase-3 beta, respectively, and > 60% of CC cases were positive for both phosphorylated glycogen synthase kinase-3 beta and YAP. Our study suggests that hepatocyte-derived tumors demonstrate a wide spectrum of tumor phenotypes, including HCC, CC, and hepatoblastoma-Like, through the combinatory effects of the oncogenic pathways and that the state of the phosphoinositide 3-kinase-AKT pathway is a key determinant of differentiation.

Critical role of Myc activation in mouse hepatocarcinogenesis induced by the activation of AKT and RAS pathways
B. Xin, M. Yamamoto, K. Fujii, T. Ooshio, X. Chen, Y. Okada, K. Watanabe, N. Miyokawa, H. Furukawa, Y. Nishikawa
Oncogene, 36, 36, 5087, 5097, 2017年09月07日
研究論文（学術雑誌）, MYC activation at modest levels has been frequently found in hepatocellular carcinoma. However, its significance in hepatocarcinogenesis has remained obscure. Here we examined the role of Myc activation in mouse liver tumours induced by hepatocytic expression of myristoylated AKT (AKT) and/or mutant HRAS V12 (HRAS) via transposon-mediated gene integration. AKT or HRAS alone required 5 months to induce liver tumours, whereas their combination generated hepatocellular carcinoma within 8 weeks. Co-introduction of AKT and HRAS induced lipid-laden preneoplastic cells that grew into nodules composed of tumour cells with or without intracytoplasmic lipid, with the latter being more proliferative and associated with spontaneous Myc expression. AKT/HRAS-induced tumorigenesis was almost completely abolished when MadMyc, a competitive Myc inhibitor, was expressed simultaneously. The Tet-On induction of MadMyc in preneoplastic cells significantly inhibited the progression of AKT/HRAS-induced tumours; its induction in transformed cells suppressed their proliferative activity with alterations in lipid metabolism and protein translation. Transposon-mediated Myc overexpression facilitated tumorigenesis by AKT or HRAS, and when it was co-introduced with AKT and HRAS, diffusely infiltrating tumours without lipid accumulation developed as early as 2 weeks. Examination of the dose-responses of Myc in the enhancement of AKT/HRAS-induced tumorigenesis revealed that a reduction to one-third retained enhancing effect but three-times greater introduction damped the process with increased apoptosis. Myc overexpression suppressed the mRNA expression of proteins involved in the synthesis of fatty acids, and when combined with HRAS introduction, it also suppressed the mRNA expression of proteins involved in their degradation. Finally, the MYC-positive human hepatocellular carcinoma was characterized by the cytoplasm devoid of lipid accumulation, prominent nucleoli and a higher proliferative activity. Our results demonstrate that in hepatocarcinogenesis induced by both activated AKT and HRAS, activation of endogenous Myc is an enhancing factor and adequate levels of Myc deregulation further facilitate the process with alterations in cellular metabolism.

Differential reactivation of fetal/neonatal genes in mouse liver tumors induced in cirrhotic and non-cirrhotic conditions
Xi Chen, Masahiro Yamamoto, Kiyonaga Fujii, Yasuharu Nagahama, Takako Ooshio, Bing Xin, Yoko Okada, Hiroyuki Furukawa, Yuji Nishikawa
CANCER SCIENCE, 106, 8, 972, 981, WILEY-BLACKWELL, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Hepatocellular carcinoma develops in either chronically injured or seemingly intact livers. To explore the tumorigenic mechanisms underlying these different conditions, we compared the mRNA expression profiles of mouse hepatocellular tumors induced by the repeated injection of CCl4 or a single diethylnitrosamine (DEN) injection using a cDNA microarray. We identified tumor-associated genes that were expressed differentially in the cirrhotic CCl4 model (H19, Igf2, Cbr3, and Krt20) and the non-cirrhotic DEN model (Tff3, Akr1c18, Gpc3, Afp, and Abcd2) as well as genes that were expressed comparably in both models (Ly6d, Slpi, Spink3, Scd2, and Cpe). The levels and patterns of mRNA expression of these genes were validated by quantitative RT-PCR analyses. Most of these genes were highly expressed in mouse livers during the fetal/neonatal periods. We also examined the mRNA expression of these genes in mouse tumors induced by thioacetamide, another cirrhotic inducer, and those that developed spontaneously in non-cirrhotic livers and found that they shared a similar expression profile as that observed in CCl4-induced and DEN-induced tumors, respectively. There was a close relationship between the expression levels of Igf2 and H19 mRNA, which were activated in the cirrhotic models. Our results show that mouse liver tumors reactivate fetal/neonatal genes, some of which are specific to cirrhotic or non-cirrhotic modes of pathogenesis.

Abundant Nucleostemin Expression Supports the Undifferentiated Properties of Germ Cell Tumors
Noriyuki Uema, Takako Ooshio, Kenichi Harada, Masako Naito, Kazuhito Naka, Takayuki Hoshii, Yuko Tadokoro, Kumiko Ohta, Mohamed A. E. Ali, Miyuki Katano, Tomoyoshi Soga, Yasuni Nakanuma, Akihiko Okuda, Atsushi Hirao
AMERICAN JOURNAL OF PATHOLOGY, 183, 2, 592, 603, ELSEVIER SCIENCE INC, 2013年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the rotes of NS in tumor growth in vivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells in vivo. NS-expressing cells exhibited higher Levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.

Nucleostemin in Injury-Induced Liver Regeneration
Haruhiko Shugo, Takako Ooshio, Masako Naito, Kazuhito Naka, Takayuki Hoshii, Yuko Tadokoro, Teruyuki Muraguchi, Akira Tamase, Noriyuki Uema, Taro Yamashita, Yasunari Nakamoto, Toshio Suda, Shuichi Kaneko, Atsushi Hirao
STEM CELLS AND DEVELOPMENT, 21, 16, 3044, 3054, MARY ANN LIEBERT INC, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）, The high regenerative capacity of liver contributes to the maintenance of its size and function when injury occurs. Partial hepatectomy induces division of mature hepatocytes to maintain liver function, whereas severe injury stimulates expansion of undifferentiated hepatic precursor cells, which supply mature cells. Although several factors reportedly function in liver regeneration, the precise mechanisms underlying regeneration remain unclear. In this study, we analyzed expression of nucleostemin (NS) during development and in injured liver by using transgenic green fluorescent protein reporter (NS-GFP Tg) mice. In neonatal liver, the hepatic precursor cells that give rise to mature hepatocytes were enriched in a cell population expressing high levels of NS. In adult liver, NS was abundantly expressed in mature hepatocytes and rapidly upregulated by partial hepatectomy. Severe liver injury promoted by a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine induced the emergence of NS-expressing ductal epithelial cells as hepatic precursor cells. NS knockdown inhibited both hepatic colony formation in vitro and proliferation of hepatocytes in vivo. These data strongly suggest that NS plays a critical role in regeneration of both hepatic precursor cells and hepatocytes in response to liver injury.

mTORC1 is essential for leukemia propagation but not stem cell self-renewal
Takayuki Hoshii, Yuko Tadokoro, Kazuhito Naka, Takako Ooshio, Teruyuki Muraguchi, Naoyuki Sugiyama, Tomoyoshi Soga, Kimi Araki, Ken-ichi Yamamura, Atsushi Hirao
JOURNAL OF CLINICAL INVESTIGATION, 122, 6, 2114, 2129, AMER SOC CLINICAL INVESTIGATION INC, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Although dysregulation of mTOR complex 1 (mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse hematopoiesis using a mouse model of conditional deletion of Raptor, an essential component of mTORC1. Raptor deficiency impaired granulocyte and B cell development but did not alter survival or proliferation of hematopoietic progenitor cells. In a mouse model of acute myeloid leukemia (AML), Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. mTORC1 did not control cell cycle or cell growth in undifferentiated AML cells in vivo. Transplantation of Raptor-deficient undifferentiated AML cells in a limiting dilution revealed that mTORC1 is essential for leukemia initiation. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. We further demonstrated that the reactivation of mTORC1 in those cells restored their leukemia-initiating capacity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells. Our findings provide mechanistic insight into how residual tumor cells circumvent anticancer therapies and drive tumor recurrence.

NKX2.2 Suppresses Self-Renewal of Glioma-Initiating Cells
Teruyuki Muraguchi, Shingo Tanaka, Daisuke Yamada, Akira Tamase, Mitsutoshi Nakada, Hideo Nakamura, Takayuki Hoshii, Takako Ooshio, Yuko Tadokoro, Kazuhito Naka, Yasushi Ino, Tomoki Todo, Jun-ichi Kuratsu, Hideyuki Saya, Jun-ichiro Hamada, Atsushi Hirao
CANCER RESEARCH, 71, 3, 1135, 1145, AMER ASSOC CANCER RESEARCH, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）, 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. Cancer Res; 71(3); 1135-45. (C) 2010 AACR.

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells
Takako Ooshio, Reiko Kobayashi, Wataru Ikeda, Muneaki Miyata, Yuri Fukumoto, Naomi Matsuzawa, Hisakazu Ogita, Yoshimi Takai
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 7, 5003, 5012, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta-and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-Delta PR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

TGF-beta-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia
Kazuhito Naka, Takayuki Hoshii, Teruyuki Muraguchi, Yuko Tadokoro, Takako Ooshio, Yukio Kondo, Shinji Nakao, Noboru Motoyama, Atsushi Hirao
NATURE, 463, 7281, 676, U111, NATURE PUBLISHING GROUP, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）, Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase(1). It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells(2-4). Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML(5-8). Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo.

Identification of tumor-initiating cells in a highly aggressive brain tumor using promoter activity of nucleostemin.
Akira Tamase, Teruyuki Muraguchi, Kazuhito Naka, Shingo Tanaka, Masashi Kinoshita, Takayuki Hoshii, Masako Ohmura, Haruhiko Shugo, Takako Ooshio, Mitsutoshi Nakada, Kazunobu Sawamoto, Masafumi Onodera, Kunio Matsumoto, Masanobu Oshima, Masahide Asano, Hideyuki Saya, Hideyuki Okano, Toshio Suda, Jun-ichiro Hamada, Atsushi Hirao
Proceedings of the National Academy of Sciences of the United States of America, 106, 40, 17163, 8, NATL ACAD SCIENCES, 2009年10月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16(Ink4a)/p19(Arf)-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP(+) cells as tumor-initiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP(+) brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity
Masako Ohmura, Kazuhito Naka, Takayuki Hoshii, Teruyuki Muraguchi, Haruhiko Shugo, Akira Tamase, Noriyuki Uema, Takako Ooshio, Fumio Arai, Keiyo Takubo, Go Nagamatsu, Isao Hamaguchi, Minoru Takagi, Masahiko Ishihara, Kazuhiro Sakurada, Hiromasa Miyaji, Toshio Suda, Atsushi Hirao
Stem Cells, 26, 12, 3237, 3246, 2008年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis. ©AlphaMed Press.

Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions
Takako Ooshio, Naoyuki Fujita, Akio Yamada, Tatsuhiro Sato, Yuichi Kitagawa, Ryoko Okamoto, Shinsuke Nakata, Ayaka Miki, Kenji Irie, Yoshimi Takai
Journal of Cell Science, 120, 14, 2352, 2365, 2007年07月15日, ［査読有り］
英語, 研究論文（学術雑誌）, Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. We have revealed that nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. Here, we studied the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 was necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promoted the association of afadin with nectin, whereas afadin was not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone was not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulated it. We describe here these novel roles of Par-3 in the formation of junctional complexes.

細胞の運動と細胞間接着の形成を促進するアファディンの二つの機能　　　　　　　　　　　　　　　
中田 信輔, 藤田 直之, 岡本 涼子, 山田 章夫, 佐藤 龍洋, 大塩 貴子, 北川 雄一, 高井 義美
日本癌学会総会記事, 65回, 241, 241, 日本癌学会, 2006年09月
日本語

Requirement of nectin, but not cadherin, for formation of claudin-based tight junctions in annexin II-knockdown MDCK cells
A. Yamada, N. Fujita, T. Sato, R. Okamoto, T. Ooshio, T. Hirota, K. Morimoto, K. Irie, Y. Takai
Oncogene, 25, 37, 5085, 5102, 2006年08月24日, ［査読有り］
英語, 研究論文（学術雑誌）, Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell-cell adhesion is not formed in MDCK cells in which annexin II, a Ca 2+- and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell-cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell-cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell-cell adhesion. These results indicate that the nectin-based cell-cell adhesion and afadin, but not the cadherin-based cell-cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions. © 2006 Nature Publishing Group All rights reserved.

Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells
Tatsuhiro Sato, Naoyuki Fujita, Akio Yamada, Takako Ooshio, Ryoko Okamoto, Kenji Irie, Yoshimi Takai
Journal of Biological Chemistry, 281, 8, 5288, 5299, 8, 2006年02月24日, ［査読有り］
英語, 研究論文（学術雑誌）, The Ca2+-independent immunoglobulin-like molecule nectin first forms cell-cell adhesion and then assembles cadherin at nectin-based cell-cell adhesion sites, resulting in the formation of adherens junctions (AJs). Afadin is a nectin- and actin filament-binding protein that connects nectin to the actin cytoskeleton. Here, we studied the roles and modes of action of nectin and afadin in the formation of AJs in cultured MDCK cells. The trans-interaction of nectin assembled E-cadherin, which associated with p120ctn, β-catenin, and α-catenin, at the nectin-based cell-cell adhesion sites in an afadin-independent manner. However, the assembled E-cadherin showed weak cell-cell adhesion activity and might be the non-trans-interacting form. This assembly was mediated by the IQGAP1-dependent actin cytoskeleton, which was organized by Cdc42 and Rac small G proteins that were activated by the action of trans-interacting nectin through c-Src and Rap1 small G protein in an afadin-independent manner. However, Rap1 bound to afadin, and this Rap1-afadin complex then interacted with p120ctn associated with non-trans-interacting E-cadherin, thereby causing the trans-interaction of E-cadherin. Thus, nectin regulates the assembly and cell-cell adhesion activity of E-cadherin through afadin, nectin signaling, and p120ctn for the formation of AJs in Madin-Darby canine kidney cells. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Common signaling pathway is used by the trans-interaction of Necl-5/Tage4/PVR/CD155 and nectin, and of nectin and nectin during the formation of cell-cell adhesion
Tatsuhiro Sato, Kenji Irie, Ryoko Okamoto, Takako Ooshio, Naoyuki Fujita, Yoshimi Takai
Cancer Science, 96, 9, 578, 589, 9, 2005年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Nectin is a Ca2+-independent Ig-like cell-cell adhesion molecule that forms homo- and hetero-trans-dimers (trans-interaction). Nectin first forms cell-cell adhesions and then recruits cadherin to the nectin-based cell-cell adhesion sites to form AJ cooperatively with cadherin. In addition, the trans-interaction of nectin and nectin induces the activation of Cdc42 and Rac small G proteins, which enhances the formation of AJ. The activation of Cdc42 and Rac by the trans-interaction of nectin and nectin is mediated by c-Src, another small G protein, Rap1, a Cdc42-GEF, FRG, and a Rac-GEF, Vav2. Necl-5/Tage4/PVR/CD155 is another Ca2+-independent Ig-like molecule, which does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, one member of the nectin family. We show here that the trans-interaction of Necl-5 and nectin-3 bidirectionally induces the activation of Cdc42 and Rac. Similarly to the activation of Cdc42 and Rac by the trans-interaction of nectin and nectin, the trans-interaction of Necl-5 and nectin-3 first recruits and activates c-Src at the Necl-5/nectin-3-based cell-cell contact sites. c-Src then phosphorylates FRG and Vav2, and the tyrosine-phosphorylated FRG and Vav2 are recruited to the Necl-5/nectin-3-based cell-cell contact sites. The trans-interaction of Necl-5 and nectin-3 also activates Rap1 through C3G, a Rap-GEF, and this activation of Rap1 is required for the activation of Cdc42 and Rac. These results indicate that the trans-interactions of Necl-5 and nectin-3 and of nectin and nectin induce the activation of Cdc42 and Rac through the common signaling molecules c-Src, Rap1, FRG, and Vav2. © Japanese Cancer Association.

Involvement of the annexin II-S100A10 complex in the formation of E-cadherin-based adherens junctions in madin-darby canine kidney cells
Akio Yamada, Kenji Irie, Takeshi Hirota, Takako Ooshio, Atsunori Fukuhara, Yoshimi Takai
Journal of Biological Chemistry, 280, 7, 6016, 6027, 2005年02月18日, ［査読有り］
英語, 研究論文（学術雑誌）, E-cadhetin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca 2+, E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca2+, E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca2+, E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca2+ in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca2+, E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-eadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the Bectin-based cell-cell contact sites in the Ca2+ switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.

Involvement of heterophilic trans-interaction of Necl-5/Tage4/PVR/CD155 with nectin-3 in formation of nectin- and cadherin-based adherens junctions
Tatsuhiro Sato, Kenji Irie, Takako Ooshio, Wataru Ikeda, Yoshimi Takai
Genes to Cells, 9, 9, 791, 799, 9, 2004年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Nectins, Ca2+ -independent immunoglobulin (Ig)-like cell-cell adhesion molecules and cadherins, Ca2+ -dependent cell-cell adhesion molecules, are associated through their respective cytoplasmic tail-binding proteins, afadin and catenins and play roles in formation of adherens junctions (AJs) in epithelial cells and fibroblasts. Nectin-like molecule-5 (Necl-5) is a Ca2+ -independent Ig-like molecule which does not homophilically trans-interact, but heterophilically trans-interacts with nectin-3, one member of the nectin family. Necl-5 does not directly bind afadin and therefore is not associated with cadherins. Necl-5 regulates cell motility and proliferation in cooperation with integrins and growth factor receptors, when it does not interact with nectin-3. We studied here a role of the heterophilic trans-interaction of Necl-5 with nectin-3 in cell-cell adhesion using L cells stably expressing Necl-5, nectin-3 and E-cadherin (Necl-5-nectin-3-EL cells). Afadin, E-cadherin and catenins were recruited to the nectin-3 side, but not to the Necl-5 side, of the contact sites formed by the heterophilic trans-interaction between Necl-5 and nectin-3. The anti-Necl-5 monoclonal antibody, which specifically inhibited the heterophilic trans-interaction of Necl-5 with nectin-3, inhibited the formation of the E-cadherin-based AJs in Necl-5-nectin-3-EL cells. These results indicate that Necl-5 plays roles not only in cell motility and proliferation but also in cell-cell adhesion in cooperation with nectin-3. © Blackwell Publishing Limited.

Requirement of the actin cytoskeleton for the association of nectins with other cell adhesion molecules at adherens and tight junctions in MDCK cells
Akio Yamada, Kenji Irie, Atsunori Fukuhara, Takako Ooshio, Yoshimi Takai
Genes to Cells, 9, 9, 843, 855, 2004年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Nectins, Ca2+-independent immunoglobulin-like cell adhesion molecules (CAMs), first form cell-cell adhesion where cadherins are recruited, forming adherens junctions (AJs) in epithelial cells and fibroblasts. In addition, nectins recruit claudins, occludin, and junctional adhesion molecules (JAMs) to the apical side of AJs, forming tight junctions (TJs) in epithelial cells. Nectins are associated with these CAMs through peripheral membrane proteins (PMPs), many of which are actin filament-binding proteins. We examined here the roles of the actin cytoskeleton in the association of nectins with other CAMs in MDCK cells stably expressing exogenous nectin-1. The nectin-1-based cell-cell adhesion was formed and maintained irrespective of the presence and absence of the actin filament-disrupting agents, such as cytochalasin D and latrunculin A. In the presence of these agents, only afadin remained at the nectin-1-based cell-cell adhesion sites, whereas E-cadherin and other PMPs at AJs, α-catenin, β-catenin, vinculin, α -actinin, ADIP, and LMO7, were not concentrated there. The CAMs at TJs, claudin-1, occludin and JAM-1, or the PMPs at TJs, ZO-1 and MAGI-1, were not concentrated there, either. These results indicate that the actin cytoskeleton is required for the association of the nectin-afadin unit with other CAMs and PMPs at AJs and TJs. © Blackwell Publishing Limited.

Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and α-actinin in epithelial cells
Ooshio T, Irie K, Morimoto K, Fukuhara A, Imai T, Takai Y
Journal of Biological Chemistry, 279, 30, 31365, 31373, 2004年07月23日, ［査読有り］
英語, 研究論文（学術雑誌）, Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that are involved in formation of cadherin-based adherens junctions (AJs). The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually enhances the formation of AJs through reorganization of the actin cytoskeleton. Although evidence has accumulated that nectins recruit cadherins to the nectin-based cell-cell adhesion sites through their cytoplasm-associated proteins, afadin and catenins, it is not fully understood how nectins are physically associated with cadherins. Here we identified a rat counterpart of the human LIM domain only 7 (LMO7) as an afadin- and α-actinin-binding protein. Rat LMO7 has two splice variants, LMO7a and LMO7b, consisting of 1,729 and 1,395 amino acids, respectively. LMO7 has calponin homology, PDZ, and LIM domains. Western blotting revealed that LMO7 was expressed ubiquitously in various rat tissues. Immunofluorescence and immunoelectron microscopy revealed that LMO7 localized at cell-cell AJs, where afadin localized, in epithelial cells of rat gallbladder. In addition, LMO7 localized at the cytoplasmic faces of apical membranes in the same epithelial cells. We furthermore revealed that LMO7 bound α-actinin, an actin filament-bundling protein, which bound to α-catenin. Immunoprecipitation analysis revealed that LMO7 was associated with both the nectin-afadin and E-cadherin-catenin systems. LMO7 was assembled at the cell-cell adhesion sites after both the nectin-afadin and E-cadherin-catenin systems had been assembled. These results indicate that LMO7 is an afadin- and α-actinin-binding protein that connects the nectin-afadin and E-cadherin-catenin systems through α-actinin.

ショウジョウバエを活用したがん研究の総説論文を発表～新規がん治療法の開発に期待～　　　　　　　　　　　　　　　
山村凌大, 大塩 貴子, 園下将大, 北海道大学・プレスリリース, 2021年07月, ［筆頭著者］

肝傷害後の組織修復におけるストレス関連キナーゼMKK7の役割　　　　　　　　　　　　　　　
大塩 貴子, 西川祐司, 肝細胞研究会・研究交流, 2021年05月, ［招待有り］, ［筆頭著者］

Myc非依存性の肝細胞再生性増殖におけるプロリン代謝の意義　　　　　　　　　　　　　　　
後藤 正憲, 大塩 貴子, 山本 雅大, 人見 淳一, 藤井 裕美子, 田中 宏樹, 上小倉 佑機, 孟 玲童, 岡田 陽子, 西川 祐司, 日本癌学会総会記事, 79回, OJ13, 4, 2020年10月
(一社)日本癌学会, 英語

肝細胞のストレスキナーゼ MKK7 は傷害後の肝組織修復に関与する　　　　　　　　　　　　　　　
Takako Ooshio, Masahiro Yamamoto, Kiyonaga Fujii, Bing Xin, Kenji Watanabe, Masanori Goto, Yoko Okada, Akira Suzuki, Josef M. Penninger, Hiroshi Nishina, Yuji Nishikawa, 旭川医科大学・プレスリリース, 2020年10月, ［筆頭著者］

Myc is involved in DNA synthesis, but not in glycometabolic changes of cultured mouse hepatocytes
Masanori Goto, Takako Ooshio, Kiyonaga Fujii, Masahiro Yamamoto, Kenji Watanabe, Bing Xin, Yoko Okada, Yuji Nishikawa, CANCER SCIENCE, 109, 456, 456, 2018年12月
WILEY, 英語, 研究発表ペーパー・要旨（国際会議）

肝細胞の再生性および腫瘍性増殖におけるMKK7の機能解析 (平成24・25年度「独創性のある生命科学研究」個別研究課題)
大塩 貴子, 旭川医科大学研究フォーラム = Asahikawa Medical University research bulletin, 15, 62, 64, 2014年
雑誌掲載版, 旭川医科大学, 日本語

タイトジャンクション形成におけるアファディンとZO‐1の相互作用の解析
小林礼子, 大塩貴子, 池田わたる, 宮田宗明, 扇田久和, 高井義美, 生化学, ROMBUNNO.2P-713, 2009年09月25日
日本語

Identifying RFK and MEK as potential therapeutic targets for pancreatic cancer through a Drosophila screening　　　　　　　　　　　　　　　
Ooshio T, Sonoshita M
6th Edition of International Cancer Conference, 2023年08月19日, 英語
2023年08月17日 - 2023年08月19日

Identifying a novel therapeutic candidate for pancreatic cancer using a Drosophila model　　　　　　　　　　　　　　　
Ooshio T, Satoh Y, Fujii K, Fukuda J, Ishihara T, Sonoshita M
Serendipity Workshop 2022, 2022年05月14日, 英語, ポスター発表
2022年05月14日 - 2022年05月14日

肝細胞から胆管細胞への分化転換におけるNotch経路と上皮間葉転換の関与　　　　　　　　　　　　　　　
大塩 貴子
第108回日本病理学会総会, 2019年05月, 口頭発表（一般）

肝細胞の上皮間葉転換：胆管上皮細胞への分化転換における相反的意義　　　　　　　　　　　　　　　
大塩 貴子
第51回北海道病理談話会, 2018年10月, 口頭発表（一般）

肝細胞から胆管上皮細胞への分化転換における上皮間葉転換の関与　　　　　　　　　　　　　　　
大塩 貴子
第107回日本病理学会総会, 2018年07月, 口頭発表（一般）

肝細胞から胆管上皮細胞への分化転換に一過性の上皮間葉転換 (EMT) が先行する　　　　　　　　　　　　　　　
大塩 貴子
第25回肝細胞研究会, 2018年06月, 口頭発表（一般）

肝細胞の胆管上皮細胞への分化転換における上皮間葉転換の意義　　　　　　　　　　　　　　　
大塩 貴子
第28回日本肝臓医生物学研究会, 2018年01月, 口頭発表（一般）

肝細胞から胆管上皮細胞への分化転換における上皮間葉転換の関与　　　　　　　　　　　　　　　
大塩 貴子
第50回北海道病理談話会, 2017年10月, 口頭発表（一般）

炎症性サイトカインによる肝細胞のSAA発現誘導へのストレスキナーゼMKK7の関与　　　　　　　　　　　　　　　
大塩 貴子
第24回肝細胞研究会, 2017年07月, ポスター発表

肝組織修復におけるストレスキナーゼMKK7の役割：マトリックスを介した肝細胞移動能への影響について　　　　　　　　　　　　　　　
大塩 貴子
第106回日本病理学会総会, 2017年04月, ポスター発表

肝細胞MKK7によるplasminogen活性化因子の発現制御：急性肝障害における病態生理学的意義　　　　　　　　　　　　　　　
大塩 貴子
第39回日本分子生物学会年会, 2016年12月, 口頭発表（一般）

肝細胞におけるストレスキナーゼMKK7の役割：初代培養肝細胞を用いた検討　　　　　　　　　　　　　　　
大塩 貴子
第23回肝細胞研究会, 2016年07月, ポスター発表

肝細胞の増殖および三次元形態形成におけるストレスキナーゼMKK7の役割　　　　　　　　　　　　　　　
大塩 貴子
第105回日本病理学会総会, 2016年05月, ポスター発表

ストレスキナーゼMKK7の肝組織リモデリングへの関与　　　　　　　　　　　　　　　
大塩 貴子
BMB, 2015年12月, 口頭発表（一般）

ストレスキナーゼMKK7の肝組織リモデリングへの関与　　　　　　　　　　　　　　　
大塩 貴子
第48回北海道病理談話会, 2015年10月, 口頭発表（一般）

肝障害後の組織修復におけるストレス関連キナーゼMKK7の役割　　　　　　　　　　　　　　　
大塩 貴子
第104回日本病理学会総会, 2015年05月, ポスター発表

オートファジーを標的とする膵がんの新規治療法の開発
科学研究費助成事業
2023年04月 - 2026年03月
大塩 貴子, 園下 将大, 野田 展生
日本学術振興会, 基盤研究(C), 北海道大学, 23K06667

膵がん細胞においてリボフラビン経路およびMEKが制御する代謝経路の同定　　　　　　　　　　　　　　　
2023年度女性研究者リーダー育成共同研究助成
2023年09月 - 2024年03月
北海道大学, 研究代表者

膵臓がんの頑健性の分子基盤の解明とその破壊による新規治療法の確立
科学研究費助成事業 基盤研究(B)
2020年04月01日 - 2023年03月31日
園下 将大, 藤井 清永, 市川 聡, 大塩 貴子, 小沼 剛
がんは日本人の死因の第一位で、対策が急務となっている。種々のがんの中でも特に治療の選択肢が少なく、患者の予後が最も悪いものが膵がんである。哺乳類実験系で膵がんの本態の解明や治療標的候補の同定が進められてきたものの、それらの知識はいまだ断片的で、詳細な発生機序や、他のがん種に比べて治療に対する頑健性が極めて高い理由は解明されていない。
そこで本研究では、治療に対する膵がんの頑健性の成立機序を解明し、その知見に基づいて新規膵がん治療戦略を創出する。特に本研究は、脆弱性を人為的に誘導する新しい方法論の提唱を目指す。本研究の遂行により、膵がんの治療応答性の詳細な分子機序を明らかにして新規膵がん治療法を確立し、膵がんの本態解明と福祉向上の両面に貢献することを目指す。
我々はこれまでに、膵がん患者の中で最も予後不良の患者群が有する4遺伝子変異(がん遺伝子KRASの活性化、がん抑制遺伝子群TP53やCDKN2A、SMAD4の不活性化)を模倣したハエを作出することに成功している。そして、このハエを使用してキナーゼの網羅的解析を実施し、膵がんの新規治療標的となるキナーゼ群を同定した。我々はこれらを阻害する化合物も見出したが、臨床と同様、効果は限定的だった。
そこで我々は本年度、この頑健性に関わるシグナル経路の同定に取り組んだ。すなわち、この化合物を上記ハエに投与した状態で網羅的遺伝学スクリーニングを実施した。その結果、阻害によりこの化合物の抗腫瘍効果を向上させるキナーゼが存在することを突き止めた。この結果は、このキナーゼが当該化合物に対する膵がんの頑健性の成立に重要な役割を果たしていることを示唆している。
日本学術振興会, 基盤研究(B), 北海道大学, 20H03524

リボフラビン経路を標的とした新規膵臓がん治療法の開発
科学研究費助成事業 基盤研究(C)
2020年04月01日 - 2023年03月31日
大塩 貴子, 園下 将大, 市川 聡, 藤井 清永
日本学術振興会, 基盤研究(C), 北海道大学, 20K07558

オートファジー阻害を起点とした新規膵がん治療法の開発　　　　　　　　　　　　　　　
2022年度 研究助成（一般）
2022年08月 - 2023年03月
大塩貴子
公益財団法人 秋山記念生命科学振興財団, 北海道大学, 研究代表者

膵がん形質に影響を与えるビタミンB群の同定とその知見に基づいた新規膵がん治療法の開発　　　　　　　　　　　　　　　
第36回（令和３年度）寿原記念財団研究助成
2022年04月 - 2023年03月
大塩貴子
公益財団法人 寿原記念財団, 北海道大学, 研究代表者

リボフラビン類縁体に立脚した新規膵がん治療法の開発　　　　　　　　　　　　　　　
令和３年度 次世代研究者リーダー育成共同研究助成
2021年09月 - 2022年03月
大塩貴子
北海道大学, 北海道大学, 研究代表者

膵がんでリボフラビン経路が調節する代謝経路とその分子機序の同定　　　　　　　　　　　　　　　
2020年度医学系研究助成
2020年10月 - 2021年03月
大塩 貴子
公益財団法人武田科学振興財団, 研究代表者

リボフラビン経路による膵臓がん形質の促進機構の解明　　　　　　　　　　　　　　　
2020年度研究助成金
2020年08月 - 2021年03月
大塩 貴子
公益財団法人日本応用酵素協会, 研究代表者, 競争的資金

遺伝子変異の多様性が膵臓がん形質に及ぼす影響の解明と新規膵臓がん治療法の開発　　　　　　　　　　　　　　　
2019年度がん研究助成
2020年04月 - 2021年03月
園下 将大、大塩 貴子
公益財団法人がん研究振興財団, 研究分担者

リボフラビン類縁体の創生に立脚した新規膵臓がん治療法の開発　　　　　　　　　　　　　　　
北海道大学病院臨床研究開発センター橋渡し研究戦略的推進プログラム2020年度シーズA支援
2020年04月 - 2021年03月
大塩 貴子, 園下 将大, 市川 聡, 関谷 翔, 佐藤 悠介, 藤井 清永, 小沼 剛
AMED, 研究代表者

遺伝子変異の多様性が膵臓がん発生に及ぼす影響の解明と新規膵臓がん治療法の開発　　　　　　　　　　　　　　　
第34回研究助成
2019年12月 - 2021年03月
園下 将大、大塩 貴子
公益財団法人寿原記念財団, 研究分担者

膵臓がんの薬物治療抵抗性の克服に立脚した 新規治療法の開発　　　　　　　　　　　　　　　
2019年度がん領域スタートアップ研究助成
2020年01月 - 2020年12月
園下 将大、大塩 貴子
公益財団法人MSD生命科学財団, 研究分担者

膵臓がん発生を促進する代謝経路の同定とそれに立脚した新規治療法の開発　　　　　　　　　　　　　　　
令和元年度研究助成
2019年12月 - 2020年12月
園下 将大、大塩 貴子
公益財団法人鈴木謙三記念医科学応用研究財団, 研究分担者

腸内細菌叢が膵臓がんの形成と薬物応答 に及ぼす影響の解明とそれに立脚した新 規膵臓がん治療戦略の確立　　　　　　　　　　　　　　　
2019年度研究助成金
2019年12月 - 2020年12月
園下 将大、大塩 貴子
公益財団法人持田記念医学薬学振興財団, 研究分担者

リボフラビン代謝経路による膵臓がん発生促進機序の解明と新規膵臓がん　　　　　　　　　　　　　　　
2019年度生命科学研究助成
2019年11月 - 2020年12月
園下将大
公益財団法人武田科学振興財団, 研究分担者

生体内でのビタミン機能の網羅的な解析基盤の創出〜モデル動物の利用と農産物の解析技術の融合〜　　　　　　　　　　　　　　　
部局横断型若手研究助成事業共同研究助成
2019年11月 - 2020年03月
大塩 貴子、星野 洋一郎
国立大学法人北海道大学, 研究代表者, 競争的資金

腸内細菌叢が膵臓がんの発生に与える影響の解明とそれに立脚した新規治療法の開発　　　　　　　　　　　　　　　
がん進展制御研究所「共同研究」
2019年04月 - 2020年03月
園下将大
国立大学法人金沢大学, 研究分担者

ホットスポット変異型RAS群ががん発生を促進する機序の差異の解明と新規治療法の開発　　　　　　　　　　　　　　　
2019年度研究助成金
2019年04月 - 2020年03月
園下将大
公益財団法人日本応用酵素協会, 研究分担者

免疫チェックポイントを阻害する新規化合物の探索　　　　　　　　　　　　　　　
酵素学研究所「共同研究」
2019年04月 - 2020年03月
園下 将大、大塩 貴子
国立大学法人徳島大学, 研究分担者

肝上皮系細胞の分化を規定するマスタースイッチの探索
科学研究費助成事業 挑戦的萌芽研究
2015年04月01日 - 2017年03月31日
西川 祐司, 大塩 貴子, 藤井 清永, 山本 雅大
肝細胞から胆管上皮細胞の分化転換に伴い，転写因子HNF-4αのP1アイソフォームからP2アイソフォームへの切り替えが認められるが，その詳細な機構は不明である．P1プロモーターは上流転写因子のCOUP-TF，CTIPなどに制御されていることが報告されているが，これらの発現増加は弱く，P1アイソフォームの発現減少にやや遅れて生じるため，アイソフォーム切り替えの原因ではないことが示唆された．一方，Notch阻害剤DAPTやNotch intracellular domain発現ベクター導入により，NotchシグナリングがP2プロモーター活性化と肝細胞の分化転換に重要であることが明らかになった．
日本学術振興会, 挑戦的萌芽研究, 旭川医科大学, 15K15107

肝再生および肝腫瘍形成におけるJNK経路の意義とその制御機構
科学研究費助成事業 若手研究(B)
2014年04月01日 - 2017年03月31日
大塩 貴子, 西川 祐司
JNKの上流キナーゼMKK7は肝細胞増殖や肝再生への関与が示唆されているが，不明な点は多い．我々は肝特異的MKK7ノックアウト (KO) マウスを用い,MKK7 KOは四塩化炭素投与による傷害の程度や肝細胞増殖に影響を与えないが，組織修復を遅延させることを見出した．初代培養肝細胞をコラーゲンゲル3次元培養したところ，MKK7 KOでは樹枝状形態形成が抑制され，transgelinとplasminogen活性化因子の発現が低下した．これらの遺伝子をMKK7 KO肝細胞に過剰発現させると，形態形成能が回復した．以上より，MKK7は肝細胞の運動に関与し，肝修復を促進することが示唆された．
日本学術振興会, 若手研究(B), 旭川医科大学, 26860255

肝細胞と細胆管上皮の相互可塑性―細胆管反応における意義の解明と肝疾患治療への展開
科学研究費助成事業 基盤研究(B)
2012年04月01日 - 2016年03月31日
西川 祐司, 山本 雅大, 藤井 清永, 大塩 貴子, 丸山 弘樹
慢性肝傷害に伴い高頻度に認められる胆管増加（細胆管反応）のメカニズムをin vivoにおける肝細胞追跡系を用いて検討した．その結果，傷害部において成熟肝細胞から細胆管上皮細胞への分化転換（化生）が起こることが証明された．また，小葉中心性慢性肝傷害においては，傷害部での細胆管増加は肝細胞の脱分化に加え，グリソン鞘周囲の細胆管および胆管の増殖および傷害部への移動（リモデリング）が起こることが示された．我々の結果は，細胆管反応が既存の肝細胞および胆管上皮細胞の分化・増殖異常に基づくものであることを示唆しており，慢性肝疾患の治療戦略を考える上で重要な知見を提供するものである．
日本学術振興会, 基盤研究(B), 旭川医科大学, 24390092

プリンヌクレオチド合成経路によるがん幹細胞の未分化維持機構の解明
科学研究費助成事業 特別研究員奨励費
2010年 - 2012年
大塩 貴子
申請者のグループでは、幹細胞マーカー候補のNucleostemin(NS)の発現を指標に、がん幹細胞および正常組織幹細胞の特定と未分化性維持機構の解明に取り組んでいる。これまでに、精巣、肝臓および脳腫瘍におけるNS高発現細胞には、正常組織幹細胞もしくはがん幹細胞が濃縮していることを報告しており、NSは組織を越えた未分化マーカーとなりうることが示唆されている。本年度は、マウスおよびヒトにおける精巣、胚細胞腫瘍でのNSの発現を解析した。マウスおよびヒトの胎児精巣、ヒト胚細胞腫瘍でのNSの発現を調べたところ、NS陽性細胞の約60%が未分化マーカーであるOct3/4陽性であった。これらの結果より、NSは精巣および胚細胞腫瘍での幹細胞マーカーであることが示唆された。さらに胚細胞腫瘍でのNS発現細胞の機能を検討するために、マウス奇形腫モデルを用いて解析を行った。マウスES細胞はNS強陽性だが、ES細胞を免疫不全マウスの皮下に移植し奇形腫を作らせると、ES細胞の殆どはNS陰性および弱陽性になった。免疫組織染色により、NS陽性細胞は分化マーカー陰性であり、未分化マーカーや増殖マーカー陽性であった。奇形腫よりNS強陽性、弱陽性、陰性細胞を分離し、in vitroでの培養と免疫不全マウスへの2次移植を行ったところ、NS強陽性細胞は高いコロニー形成能および腫瘍形成能を保持しており、奇形腫においてもNS強発現細胞集団には、がん幹細胞が濃縮していることが明らかとなった。さらに奇形腫においてNSをノックアウトすると、腫瘍の形成は優位に抑えられ、未分化マーカー陽性細胞が増加していた。以上の結果より、NSは奇形腫において未分化維持や細胞増殖に寄与していることが判明した。今後、効率よくNSの発現を抑制できる薬剤が開発されれば、胚細胞腫瘍においてがん幹細胞を分化させ、がんの根治に繋がるものと期待される。
日本学術振興会, 特別研究員奨励費, 金沢大学, 10J06332

ネクチン-アファディン系による細胞間接着の形成と破壊の分子機構
科学研究費助成事業 特別研究員奨励費
2005年 - 2007年
大塩 貴子
多細胞生物において細胞間接着は、発生過程における組織構築や正常組織の維持など、多くの生命現象に重要な細胞機能である。私共は、上皮細胞において接着分子ネクチンがまず細胞間接着を形成し、低分子量Gタンパク質Cdc42とRacを活性化してアクチン細胞骨格を形成させること、さらにその部位に接着分子カドヘリンをリクルートしてアドヘレンスジャンクション(AJ)、タイトジャンクション(TJ)、および細胞極性を形成することを明らかにしている。本研究では、ネクチン-アファディン系によるAJとTJ形成の分子機構を解析し、以下の結果を得た。
(1)細胞同士が接着を作るとき、まずネクチンがトランス結合し、その細胞内でアファディンが結合するのにPar-3、Par-6、aPKC複合体が必要であった。
(2)ネクチンのトランス結合によるRacの活性化には、Par-3が必要でなかった。
3)アファディンがネクチンによって活性化されたRapに結合することにより、p120カテニンを介してカドヘリンをトランス結合させるのに、Par-3が必要であった。
(4)カドヘリンのトランス結合による、Racの活性化にもPar-3は必要だった。
(5)アファディンがTJ分子をAJのアピカル側にリクルートするのにも、Par-3は必要だった。
このように、ネクチン-アファディン系は、Par-3と強調的にAJとTJを形成していることが明らかになった。
日本学術振興会, 特別研究員奨励費, 大阪大学, 05J09953

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市販のペットボトルのお茶に含まれるビタミンCの定量　　　　　　　　　　　　　　　
2017年12月
講師
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旭川医科大学派遣講座No.31「生命科学実習〜ヒトの体のしくみを知ろう」

マウスの解剖　　　　　　　　　　　　　　　
2016年08月
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動物学会北海道支部会