Our long-term goal is to understand the molecular mechanisms which coordinatelyregulate growth and differentiation of stem cells as well as differentiated cells withemphasis on intracellular signal transduction. For this purpose we are using mod-els ranging from iPS and various culture cells, zebrafish, mouse, to monkey.Based on our research background on the area of cytokine signals, we now focuson the analysis of development and regeneration of neural retina.

The neural retina is a part of the central nerv-ous system (CNS), and regeneration of the ret-ina from retinal stem cells or other sources bytransplantation is a critical issue from both clini-cal and neurobiological points of view. Al-though reports of successful regeneration of theCNS from neural stem cells (NSC) have ap-peared in the literature, such has not been thecase for the vertebrate neural retina. Further-more, the nature of retinal stem cells has notbeen clarified, making it difficult to attempt re-generation of the retina. Based on the tech-niquears and knowledge that have been accu-mulated through work on haematopoietic sys-tems in our laboratory, we attempt to identifymammalian retinal stem cells and following de-velopmental processes by revealing the expres-sion pattern of cell surface proteins. We foundthat various CD antigens mark spatiotemporallydistinct populations of retinal cells, and genesspecifically expressed in such populations havebeen revealed by microarray analyses. Varioussignaling molecules and transcriptional factorsare under investigation for their roles on retinaldevelopment. For developmental biologicalanalyses, we use zebrafish in addition to mouseas model animals. We also continue to work on

haematological projects, and bidirectional coop-erative progress between neurological and hae-matological works is one of unique features ofour laboratory. Projects, which gave major find-ings during 2011 are as follows.

1. Serine/Threonine kinase Melk regulatesproliferation and glial differentiation of reti-nal progenitor cells

Rika Saito, Sumiko Watanabe

Serine/threonine kinase, Melk, was initiallycloned in oocytes, but it is expressed in normaltissues and especially in cancer cells. We previ-ously identified Melk as a gene that is highlyexpressed in immature mouse retinal progeni-tors. To analyze the function of Melk in embryo-genesis, we cloned zebrafish Melk and reportedthat morpholino-based downregulation of Melkin zebrafish resulted in severe anemia (Saito etal. MCB, 2005). Melk-morpholino-treated ze-brafish also showed microphthalmia, suggestingthe participation of Melk in retinal development.In Melk-depleted retinas, differentiation of reti-nal neurons took place but was delayed, and theproliferative period of retinal progenitor cells

Donatinon Laboratories

Department of Molecular and Developmental Biology再生基礎医科学寄付研究部門（SBI，トミー，ロート製薬，慈照会）

Visiting Professor Sumiko Watanabe, Ph.D.Visiting Research Associate Shinya Satoh, Ph.D.Visiting Research Associate Hideto Koso, MD, Ph.D

特任教授 医学博士 渡 辺 すみ子特任助教 工学博士 佐 藤 伸 哉特任助教 医学博士 高 祖 秀 登

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was prolonged, suggesting that Melk may regu-late the timing of the transition from prolifera-tion to differentiation. For more detailed exami-nation, we performed gain- and loss-of-functionanalyses of Melk in mouse retinas. Knockdownof Melk by shRNA in mouse embryonic retinalexplant culture resulted in decreased prolifera-tive activity of retinal progenitors, and accord-ingly, overexpression of Melk slightly enhancedproliferation. Differentiation of retinal progeni-tor into subtypes of retinal neurons was not sig-nificantly affected, but Müller glia differentia-tion was perturbed by the level of Melk. Fur-thermore, process extension of glial cells was en-hanced in the absence of Melk, suggesting thatMelk is involved in the morphological differen-tiation of retinal cells. Taken together, our re-sults suggest that Melk is primarily required forproper proliferation, and may play multipleroles in retinal development in vertebrates.

2. Identification of CD44 as a cell surfacemarker for Müller glia precursor cells

Toru Shinoe, Hiroshi Kuribayashi, MotoharuSeiki1, Hiroyuki Aburatani2, Sumiko Watan-abe: 1Division of Cancer Cell Research, Insti-tute of Medical Science, University of Tokyo,2Genome Science Division, Research Center forAdvanced Science and Technology, Universityof Tokyo.

In the retina, both neurons and glias differen-tiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid re-ceptor expressed on mature Müller glial cells inthe retina. We found that in the developingmouse retina, expression of CD44 was tran-siently observed at or around birth in a sub-population of c-kit-positive retinal progenitorcells. The level of CD44 expression is probablyregulated primarily by transcriptional level, butour results also suggested the involvement ofposttranslational regulation. During in vitro cul-ture, purified CD44/c-kit-positive retinal pro-genitor cells exclusively differentiated intoMüller glial cells and not into neurons, suggest-ing that CD44 marks a subpopulation of retinalprogenitor cells that are fated to become glias.Over-expression of CD44 inhibited the extensionof processes by Müller glial cells and neurons,and involvement of hyaluronic acid in this proc-ess was suggested. Notch signaling is known tobe involved in the specification of retinal pro-genitors into a glial fate. Activation of Notchsignaling increased the number of CD44-positivecells in early development of the retina, andtreatment with the Notch signal inhibitor,DAPT, at early, but not later, stages of retinal

development abolished both CD44-positive cellsand Müller glial cells. Together, CD44 was iden-tified as an early cell surface marker of theMüller glia lineage, and Notch signalling wasshown to be involved in commitment of retinalprogenitor cells to CD44 positive Müller glialprecursor cells.

3. The early retinal progenitor-expressinggene Sox11 regulates the timing of the dif-ferentiation of retinal cells

Ayumi Usui3, Yujin Mochizuki3, Atsumi Iida,Hiroyuki Aburatani2, Sumiko Watanabe: 3De-partment of Ophthalmology, Juntendo Univer-sity School of Medicine.

Sry-related HMG box (Sox) proteins play di-verse and critical roles in a variety of morpho-genetic processes during embryonic develop-ment. Sox11 and Sox4 are members of the SoxCsubtype. We found that Sox11 was strongly ex-pressed in early retinal progenitor cells and thatwhen expression of Sox11 subsided aroundbirth, Sox4 expression began. To analyze therole of Sox11 and Sox4 in retinal development,we perturbed their expression pattern by ex-pressing them ectopically in retinal explant cul-ture. Overexpression of Sox11 or Sox4 in retinalprogenitors resulted in similar phenotypes, thatis, increased cone cells and decreased Müllerglia. Birth-date analysis showed that cone cellswere produced at a later developmental stagethan that for normal cone genesis. Sox11-knockout retinas showed delayed onset and pro-gress of differentiation of subsets of retinal cellsduring the embryonic period. After birth, retinaldifferentiation took place relatively normally,probably because of the redundant activity ofSox4, which starts to express around birth. Nei-ther overexpression nor loss-of-function analysisgave any evidence that Sox11and Sox4 directlyregulate transcription of genes critical to differ-entiation of subsets of retinal cells. However,histone H3 acetylation status of the earlyproneural genes was lowered in knockout reti-nas, suggesting that Sox11 regulates the timingof differentiation in retinal cells by creating anepigenetic state that helps to establish the com-petency to differentiate. Taking our findings to-gether, we propose that the sequential expres-sion of Sox11 and Sox4 during retinogenesisleads to the fine adjustment of retinal differen-tiation by contributing to preparing of compe-tency of retinal progenitors.

4. Epigenetic regulation of retinal gene ex-pression during retinal development

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Shinya Satoh, Sumiko Watanabe

Trimethylation at Lys27 of Histone H3 (H3K27me3) is mediated by the polycomb repressivecomplex 2 (PRC2) which contains the catalyticenzyme, Ezh1- or Ezh2-methyltransferase. Thismodification provides the platform to recruitPRC1 and form the polycomb group (PcG) com-plex, which works as the transcriptional re-pressor. On the other hand, H3K27me3 is de-methylated by Jmjd3, Utx, or Uty demethylase,resulting in the activation of gene expression. Toclarify the role of this modification in retinalgene expression during retinal development, weperformed loss of function analysis of genes thatcatalyze this modification. When Jmjd3 expres-sion was knocked down by the shRNA systemin a retinal explant culture, expression of genesthat are important for differentiation into bipo-lar cells was decreased. To reveal the role of Ezh2 in retinal development, we analyzed the de-velopment of retinas that express the catalyti-cally inactive Ezh2. Ezh2 expressing mutant reti-nas showed reduction in the thickness of theONL and INL. RT-qPCR analysis showed stronginduction of Ink4a/Arf genes in Ezh2 expressingmutant retinas, suggesting that Ink4a/Arf geneexpression is regulated by H3K27me3 modifica-tion in retinal development. This data indicatesthat regulation of H3K27me3 modification is im-portant for development of certain subsets ofretinal cells.

5. Role of thyroid hormone receptor β2 in hu-man red opsin gene induction

Yo Tanaka, Shinya Satoh, Sumiko Watanabe

Cone photopigments, known as opsins, arepivotal elements and the first detection modulein color vision. There are several different op-sins, and their expression pattern in cone is pre-cisely regulated in transcription level. Red opsinis long wavelength opsin, and role of thyroidhormone and TRβ2 for transcriptional regulationhas been suggested by several ways, but de-tailed mechanisms including target sequence inthe enhancer of red opsin had not been re-vealed. We analyzed the requirement of the en-hancer region of red opsin by analyzing a largedifferent construct of red opsin enhancer/promoter-luciferase plasmids. We found thatTRβ2 affected 5’ and exon/intron region, but ef-fects of T3 was only observed with exon/intronregion. Furthermore, COUP-TFII suppressed theenhance/promoter activity of red opsin gene.

6. Role of Sall family transcription factors ineye development

Yukihiro Baba, Yui Watabe, Sumiko Watan-abe

The vertebrate homolog of spalt, sall, includesfour members, and Sall1 and Sall4 are involvedin normal development, particularly of limbsand the nervous system, as well as several or-gans including the kidney and heart. The spaltgenes are mutated in several human congenitalsyndromes and expressed in the developing em-bryo in discrete but overlapping patterns. Wefocus on Sall1 and Sall3 to reveal their roles inretinal development.6.1 Induction of horizontal cell differentiation in

mouse embryonic retinal cells by combinato-rial expression of Foxn4, Prox1 and Sall3

Several transcription factors such as Foxn4, Ptf1a, and Prox1 have been shown to be essentialfor horizontal cell fate determination. However,none of these factors were sufficient to inducehorizontal cells from retinal progenitor cells inmouse retina. In addition, we found that thespalt-like zinc finger protein family Sall3 was es-sential for maturation of horizontal cells, butagain it could not induce horizontal cell fate byitself. Therefore, a combinational function ofthese transcription factors might be required forhorizontal cell differentiation. To verify this hy-pothesis, gain-of-function analysis was per-formed with transcription factors in combina-tion. Various combinations of pCAG plasmidsencoding Foxn4, Ptf1a, Prox1, Lim1, and Sall3were introduced into mouse retinal explant cul-ture, and retinal differentiation and proliferationwere examined by immunostaining of frozensections. Single overexpression of any candidategenes failed to enhance the expression of hori-zontal cell markers, in agreement with earlierstudies. On the other hand, the combination ofFoxn4, Prox1, and Sall3 induced the expressionof horizontal cell markers, NF160, NFL, andCalbindin-D 28k. In addition, these cells local-ized at the appropriate position of mature hori-zontal cells. Monolayer culture confirmed hori-zontal cell-like morphology, such as long neu-rites. We concluded that horizontal cell differen-tiation is achieved by combinatorial funciton ofFoxn4, Prox1, and Sall3.6.2 Involvement of the transcriptional factor Sal-

like 1 in the lens and retinal developmentThe transcription factor Sal-like 1 is widely

expressed in the kidney, CNS, heart, limb budand anus. Homozygous Sall1 knockout is lethal,resulting in fatality 24 hours after birth from ab-sence or sever abnormalities of the kidney. Inthe eye, strong expression of Sall1 in the embry-onic lens has been reported, suggesting a poten-tial role played by Sall1 in the lens. We investi-gate the function of Sall1 in the eye, primarily

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focusing on the lens. Endogenous expression ofSall1 in the developing eye was examined usingheterozygous Sall1-EGFP mice. We found strongexpression of Sall1 in the lens. Sall1 expressionin the lens was already present at embryonicday11.5, and its expression was maintained afterbirth through to the adulthood. We also identi-fied Sall1 expression in subset of cells of retinaand the optic nerve. Morphological observationof eye revealed that many bubble-like structurein the Sall1-KO lens at embryonic day15.5. Ex-amination by electron microscopy revealed thatthese bubbles were vacuoles in the primary fibercells of the lens, and that these vacuoles ap-peared to be located between the fiber cell mem-branes. Taken together, an important role of Sall1 in the development of the lens was revealed.

7. Molecular mechanisms regulating differen-tiation and proliferation of retinal stem/pro-genitor cells

Haruna Suzuki-Kerr, Yui Watabe, SumikoWatanabe

Cell surface antigens are powerful tools forisolating specific subsets of retinal cells duringdevelopment from cell mixtures without damag-ing the cells, which makes it possible to charac-terize their properties and identify genes thatregulate their proliferation and differentiation.By screening retinal cells from mice at variousdevelopmental stages for their reactivity withover 150 different antibodies against various cellsurface antigens, we identified SSEA-1 and c-kitas early and late progenitor markers, respec-tively. SSEA-1 marks retinal progenitor cells inthe peripheral region of the retina at around E14-E16. In the later stage of embryogenesis, SSEA-1 disappears and c-kit expression is observed inthe retinal progenitor cells in the central regionof the retina. We compared the gene expressionpatterns of regionally and temporally differentsubsets of retinal progenitor cells, SSEA-1-positive cells at E14, c-kit positive cells at P1,and differentiated c-kit negative cells at P1 us-ing a microarray. We found that several genesare specifically expressed in SSEA-1 positiveearly retinal progenitor cells.7.1 Requirement of Fezf2 in Differentiation of

Cone OFF bipolar cellsWe found the Fezf2, a transcriptional re-

pressor of the Fez zinc finger family, to bestrongly expressed in SSEA-1-positive cells. Fezf2 expression pattern was characterized in themature Fezf2＋/－ retinas by making use of the β-galactosidase reporter gene expressed under theFezf2 promoter. Between postnatal day 14 andadult, Fezf2 expression was localized to a subset

of cells in the inner nuclear layer (INL). Thesecells all expressed Chx10 but were mostly nega-tive for islet1, suggesting that Fezf2 is expressedin a subset of Chx10＋/islet1-, cone OFF bipolarpopulation. However, there was no aberrantmorphological defect in the Fezf2 deficient reti-nas at P14 or P28, albeit the slight thinning ofthe INL was observed. Further examinations re-vealed that the thinning of INL was attributedto a selective reduction in Chx10＋/islet1- coneOFF bipolar cell number. Interestingly, many ofcells expressing β-gal still persisted in Fezf2 de-ficient retinas at P14 and P28, implying that theobserved loss of OFF bipolar cells was not asimple result of Fezf2-expressing cell death.Similar analysis was then repeated with P5¯P7retinas, however, neither morphology normarker (Chx10, islet1, active caspase-3) expres-sions differed between Fezf2 deficient retinasand equivalent controls, suggesting that the ob-served phenotype may start sometime after P7.In conclusion, Fezf2 is expressed in a subset ofcone OFF bipolar cells in the mature retina, andits deficiency leads to a reduction in the numberof cone OFF bipolar cells. These results implyroles played by Fezf2 in subtype specificationand/or maturation of these bipolar cells.7.2 The role of Zic family zinc finger transcrip-

tion factors in the proliferation and differen-tiation of retinal progenitor cells

Members of the Zic family of zinc finger tran-scription factors play critical roles in a variety ofdevelopmental processes. Using DNA microar-ray analysis, we found that Zics are strongly ex-pressed in SSEA-1-positive early retinal progeni-tors in the peripheral region of the mouse ret-ina. Reverse-transcription polymerase chain re-action using mRNA from the retina at variousdevelopmental stages showed that Zic1 and Zic2are expressed in the embryonic retina and thengradually disappear during retinal development.Zic3 is also expressed in the embryonic retina;its expression level slightly decreases but it isexpressed until adulthood. We overexpressedZic1, Zic2, or Zic3 in retinal progenitors at em-bryonic day 17.5 and cultured the retina as ex-plants for 2 weeks. The number of rod photore-ceptors was fewer than in the control, but noother cell types showed significant differencesbetween control and Zic overexpressing cells.The proliferation activity of normal retinal pro-genitors decreased after 5 days in culture, as ob-served in normal in vivo developmental proc-esses. However, Zic expressing retinal cells con-tinued to proliferate at days 5 and 7, suggestingthat Zics sustain the proliferation activities ofretinal progenitor cells. Since the effects of Zic1,2, and 3 are indistinguishable in terms of differ-entiation and proliferation of retinal progenitors,

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the redundant function of Zics in retinal devel- opment is suggested.

References

1. Iida, A., Shinoe, T., Baba, Y., Mano, H.,Watanabe, S., Dicer plays essential roles forretinal development by regulation of survivaland differentiation. Invest. Ophthalmol. Vis.Sci., 52, 3008-3017, 2011

2. Ouchi, Y., Baba, Y., Koso, H., Taketo, M. M.,Iwamoto, T., Aburatani, H., Watanabe, S., β-Catenin signaling regulates the timing of celldifferentiation in mouse retinal progenitorcells, Mol. Cell. Neurosci. 46, 770-780, 2011

3. Baba, Y., Iida, A., Watanabe, S., Sall3 playsessential roles in horizontal cell maturationthrough regulation of neurofilament expres-sion levels. Biochimie, 93, 1037-1046, 2011

4. Fujii, T., Tsunesumi, S., Yamaguchi, K.,Watanabe, S., Furukawa, Y., Smyd3 is re-quired for the development of cardiac andskeletal muscle in zebrafish. Plos ONE, 6, e23491, 2011

5. Watabe, Y., Baba, Y., Nakauchi, H., Mizota,A., Watanabe, S. The role of Zic family zincfinger transcription factors in the proliferation

and differentiation of retinal progenitor cells.Biochem. Biophys. Res. Commun., 415, 42-47,2011

6. Nakamura, Y., He, X., Kato, H., Wakitani, S.,Kobayashi, T., Watanabe, S., Iida, A., Tahara,H., Warman, M. L., Watanapokasin, R., Pos-tlethwait, J. H. Sox9 is upstream of micro-RNA-140 in cartilage. Appl. Biochem. Biote-chnol. 166, 64-71, 2011

7. Iguchi-Ishiguro, H., Ouchi, Y., Watanabe, S.,Numabe, Y. Analysis of syndecan-1 gene pro-moter during mouse tooth development. Ar-chives of Oral Biology, in press

8. Saito, R., Nakauchi, H., Watanabe, S., Serine/Threonine kinase Melk regulates proliferationand glial differentiation of retinal progenitorcells. Can. Sci., in press

9. Iwagawa, T., Ohuchi, S., Watanabe, S., Naka-mura, Y. Selection of RNA aptamers againstmouse embryonic stem cells. Biochimie, 94,250-257, 2012

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Tumors contain a small population of putative cancer stem cells (CSC), whichpossess unique self-renewal properties, and survive in a quiescent state for manyyears after remission and result in later relapse and metastasis. Therefore, it isconceivable that targeting CSCs will eradicate tumor-initiating cells, whereas con-ventional chemotherapies will only eradicate the bulk of a tumor.Cancer stem cells and normal tissue stem cells utilize the same self-renewal path-way. However, researchers characterize some of changes, which occur in cancerstem cells, not in normal tissue stem cells. The design of new therapeutic agentsshould be aimed at targeting these unique molecular changes.We have currently focused on studying these unique molecular changes, whichoccur in cancer stem cells, not in normal tissue stem cells. This could be a newtherapeutic target against solid tumors.

(1) IGF1-R Kinase Inhibitors

Signaling through IGF1-R in normal cellsleads to the activation of multiple intracellularpathways, mediated by the receptor-associatedtyrosine kinase domain, by PI-3 kinase and Akt,yielding growth and enhanced survival. In can-cer cells, IGF1-R plays an even more critical rolebecause it contributes to the promotion of tumorgrowth by inhibition of the apoptosis, transfor-mation, metastasis, and induction of angiogene-sis via VEGF.

Chemosurviving cancer cells are enriched inCSCs and express increased levels of IGF-1R.Depletion of IGF-1R results in reduction of CSCphenotype in gastrointestinal cancer cells.

We have shown that a number of strategiesagainst the IGF system in cancer have beneficialantitumor effects. Dominant negative mutants,siRNA, and antagonistic antibodies and small-molecule tyrosine kinase inhibitors are being

evaluated for their ability to block signalingand, hence, the survival and growth of cancercells. Similar inhibitory effects on tumor cellgrowth and metastasis are seen in vivo, in ex-perimental animal models.

(2) Zinc-finger-containing transcriptional fac-tor, Kruppel-like factor 2 (KLF2)

The Kruppel-like factor (KLF) proteins aremultitasked transcriptional regulators with anexpanding tumor suppressor function. KLF2 is amember of the KLF family of zinc-finger tran-scription factors and is involved in maintainingT-cell quiescence, regulating preadipocyte differ-entiation, endothelial cell function, lung devel-opment and the self-renewal of ES cells. Fur-thermore, KLF2 is one of the prominent mem-bers of the family because of its diminished ex-pression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic

Donation Laboratory

Division of Antibody, Vaccine & Experimental Therapy抗体・ワクチン治療寄付研究部門

Project Professor Motoharu Seiki, D.M.Sc.Project Professor Kohzoh Imai, M.D., Ph.D.Project Professor Kouhei Tsumoto, Ph.D.Project Assistant Professor Taniguchi Hiroaki, M.D., Ph.D.

特任教授（兼） 医学博士 清 木 元 治特任教授（兼） 医学博士 今 井 浩 三特任教授（兼） 工学博士 津 本 浩 平特任助教 医学博士 谷 口 博 昭

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roles.We indicate that epigenetic silencing of KLF2

occurs in cancer cells through direct transcrip-tional repression mediated by the Polycombgroup protein Enhancer of Zeste Homolog 2(EZH2). Binding of EZH2 to the 5’-end of KLF2is also associated with a gain of trimethylatedlysine 27 histone H3 and a depletion of phos-phorylated serine 2 of RNA polymerase.

Upon depletion of EZH2 by RNA interfer-ence, short hairpin RNA or use of the smallmolecule 3-Deazaneplanocin A, the expressionof KLF2 is restored. The transfection of KLF2 incells with EZH2-associated silencing showed asignificant anti-tumoral effect, both in cultureand in xenografted nude mice.

In this last setting, KLF2 transfection was alsoassociated with decreased dissemination andlower mortality rate. In EZH2-depleted cells,which characteristically have lower tumorigenic-ity, the induction of KLF2 depletion ‘rescued’partially the oncogenic phenotype, suggestingthat KLF2 repression has an important role inEZH2 oncogenesis.

Most importantly, the translation of the de-scribed results to human primary samples dem-onstrated that patients with prostate or breasttumors with low levels of KLF2 and high ex-pression of EZH2 had a shorter overall survival.

(3) PR domain-containing protein, PRDM14

PRDM have been linked to human cancers. Toexplore the role of the PR domain family genesin breast carcinogenesis, we examined the ex-pression profiles of 16 members of the PRDMgene family in a panel of breast cancer cell linesand primary breast cancer specimens usingsemiquantitative real-time PCR.

We found that PRDM14 mRNA is overex-pressed in about two thirds of breast cancers.Moreover, immunohistochemical analysisshowed that expression of PRDM14 protein isalso up-regulated. PRDM14 are known as a keytranscription factor required for the maintenanceof hESC identity and the reacquisition of pluri-potency in human somatic cells.

Introduction of PRDM14 into cancer cells en-hanced cell growth and reduced their sensitivityto chemotherapeutic drugs. Conversely, knock-down of PRDM14 by siRNA induced apoptosisin breast cancer cells and increased their sensi-tivity to chemotherapeutic drugs, suggestingthat up-regulated expression of PRDM14 mayplay an important role in the proliferation ofbreast cancer cells.

That little or no expression of PRDM14 is seenin noncancerous tissues suggests that PRDM14could be an ideal therapeutic target for the treat-ment of breast cancer.

Publications

Project Professor, Motoharu Seiki:1. Hara, T., K. Mimura, T. Abe, G. Shioi, M.

Seiki, and T. Sakamoto, Deletion of theMint3/Apba3 gene in mice abrogates macro-phage functions and increases resistance tolipopolysaccharide-induced septic shock. JBiol Chem, 286(37): p. 32542-51, 2011.

2. Hara, T., K. Mimura, M. Seiki, and T. Saka-moto, Genetic dissection of proteolytic andnon-proteolytic contributions of MT1-MMPto macrophage invasion. Biochem BiophysRes Commun, 413(2): p. 277-81, 2011

3. Hoshino, D., N. Koshikawa, and M. Seiki, Ap27(kip1)-binding protein, p27RF-Rho, pro-motes cancer metastasis via activation ofRhoA and RhoC. J Biol Chem, 286(4): p.3139-48, 2011.

4. Itoh, Y., R. Palmisano, N. Anilkumar, H.Nagase, A. Miyawaki, and M. Seiki, Dimeri-zation of MT1-MMP during cellular invasiondetected by fluorescence resonance energytransfer. Biochem J, 440(3): p. 319-26, 2011.

5. Koga, K., M. Aoki, T. Sameshima, M.Hamasaki, N. Egawa, M. Seiki, B.P. Toole, J.Suzumiya, and K. Nabeshima, Synthetic

emmprin peptides inhibit tumor cell-fibroblast interaction-stimulated upregula-tion of MMP-2 and tumor cell invasion. Int JOncol, 39(3): p. 657-64, 2011.

6. Koshikawa, N., H. Mizushima, T. Minegishi,F. Eguchi, F. Yotsumoto, K. Nabeshima, S.Miyamoto, E. Mekada, and M. Seiki, Prote-olytic activation of heparin-binding EGF-likegrowth factor by membrane-type matrixmetalloproteinase-1 in ovarian carcinomacells. Cancer Sci, 102(1): p. 111-6, 2011.

7. Nagano, M., D. Hoshino, S. Koshiba, T.Shuo, N. Koshikawa, T. Tomizawa, F. Hay-ashi, N. Tochio, T. Harada, T. Akizawa, S.Watanabe, N. Handa, M. Shirouzu, T. Ki-gawa, S. Yokoyama, and M. Seiki, ZF21 pro-tein, a regulator of the disassembly of focaladhesions and cancer metastasis, contains anovel noncanonical pleckstrin homology do-main. J Biol Chem, 286(36): p. 31598-609,2011.

8. Nagano, M., D. Hoshino, T. Sakamoto, T.Akizawa, N. Koshikawa, and M. Seiki, ZF21is a new regulator of focal adhesion disas-sembly and a potential member of the

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spreading initiation center. Cell Adh Migr, 5(1): p. 23-8, 2011.

9. Onozuka, I., S. Kakinuma, A. Kamiya, M.Miyoshi, N. Sakamoto, K. Kiyohashi, T.Watanabe, Y. Funaoka, M. Ueyama, M.Nakagawa, N. Koshikawa, M. Seiki, H.Nakauchi, and M. Watanabe, Cholestaticliver fibrosis and toxin-induced fibrosis areexacerbated in matrix metalloproteinase-2deficient mice. Biochem Biophys Res Com-mun, 406(1): p. 134-40, 2011.

10. Saitou, T., M. Rouzimaimaiti, N. Koshikawa,M. Seiki, K. Ichikawa, and T. Suzuki, Mathe-matical modeling of invadopodia formation.J Theor Biol, 298: p. 138-46, 2011.

11. Sakamoto, T., D. Niiya, and M. Seiki, Target-ing the Warburg effect that arises in tumorcells expressing membrane type-1 matrixmetalloproteinase. J Biol Chem, 286(16): p.14691-704, 2011.

12. Shirakabe, K., S. Hattori, M. Seiki, S. Ko-yasu, and Y. Okada, VIP36 protein is a tar-get of ectodomain shedding and regulatesphagocytosis in macrophage Raw 264.7 cells.J Biol Chem, 286(50): p. 43154-63, 2011.

13. Srichai, M.B., H. Colleta, L. Gewin, L. Matri-sian, T.W. Abel, N. Koshikawa, M. Seiki, A.Pozzi, R.C. Harris, and R. Zent, Membrane-type 4 matrix metalloproteinase (MT4-MMP)modulates water homeostasis in mice. PLoSOne, 6(2): p. e17099, 2011.

14. Uematsu, T., C. Konishi, D. Hoshino, X.Han, Taizotomari, N. Egawa, Y. Takada, T.Isobe, M. Seiki, and N. Koshikawa, Identifi-cation of proteins that associate with in-tegrin alpha2 by proteomic analysis in hu-man fibrosarcoma HT-1080 cells. J CellPhysiol, 2011.

Project Professor, Kohzoh Imai:1. Nishioka M, Ueno K, Hazama S, Okada T,

Sakai K, Suehiro Y, Okayama N, Hirata H,Oka M, Imai K, Dahiya R, Hinoda Y. Possi-ble involvement of Wnt11 in colorectal can-cer progression. Mol Carcinog, 2011, inpress.

2. Ohashi H, Adachi Y, Yamamoto H, Tani-guchi H, Nosho K, Suzuki H, Arimura Y,Imai K, Carbone DP, Shinomura Y. Insulin-like growth factor receptor expression is as-sociated with aggressive phenotypes andhas therapeutic activity in biliary tract can-cers. Cancer Sci, 2011, in press.

3. Yamamoto M, Tabeya T, Naishiro Y, YajimaH, Ishigami K, Shimizu Y, Obara M, SuzukiC, Yamashita K, Yamamoto H, Hayashi T,Sasaki S, Sugaya T, Ishida T, Takano KI,Himi T, Suzuki Y, Nishimoto N, Honda S,Takahashi H, Imai K, Shinomura Y. Value ofserum IgG4 in the diagnosis of IgG4-related

disease and in differentiation from rheu-matic diseases and other diseases. ModRheumatol, 2011, in press.

4. Yamamoto M, Takahashi H, Tabeya T, Suzu-ki C, Naishiro Y, Ishigami K, Yajima H,Shimizu Y, Obara M, Yamamoto H, Himi T,Imai K, Shinomura Y. Risk of malignanciesin IgG4-related disease. Mod Rheumatol,2011, in press.

5. Liu Y, Sakamoto H, Adachi M, Zhao S, UkaiW, Hashimoto E, Hareyama M, Ishida T,Imai K, Shinomura Y. Heat stress activatesER stress signals which suppress the heatshock response, an effect occurring preferen-tially in the cortex in rats. Mol Biol Rep,2011, in press.

6. Yamamoto M, Tabeya T, Suzuki C, NaishiroY, Yajima H, Shimizu Y, Obara M, Yama-moto H, Sugaya T, Takahashi H, Imai K,Shinomura Y. Adult-onset Still’s disease inpregnancy. Mod Rheumatol, 2011, in press.

7. Fox BA, Schendel DJ, Butterfield LH, Aam-dal S, Allison JP, Ascierto PA, Atkins MB,Bartunkova J, Bergmann L, Berinstein N, Bo-norino CC, Borden E, Bramson JL, BrittenCM, Cao X, Carson WE, Chang AE, Chara-ciejus D, Choudhury AR, Coukos G, deGruijl T, Dillman R, Dolstra H, Dranoff G,Durrant LG, Finke JH, Galon J, Gollob JA,Gouttefangeas C, Grizzi F, Guida M,Hakansson L, Hege K, Herberman RB, HodiFS, Hoos A, Huber C, Hwu P, Imai K, JaffeeEM, Janetzki S, June CH, Kalinski PA, Kauf-man HL, Kawakami K, Kawakami Y, Keil-holtz U, Khleif SN, Kiessling R, Kotlan B,Kroemer G, Lapointe R, Levitsky HI, LotzeMT, Maccalli C, Maio M, Marschner J-P,Mastrangelo MJ, Masucci G, Melero I, NeliefC, Murphy WJ, Nelson B, Nicolini A,Nishimura MI, Odunsi K, Ohashi PS,O’Donnell-Tormey J, Old LJ, Ottensmeier C,Papamichail M, Parmiani G, Pawelec G,Proietti E, Qin S, Rees R, Ribas A, Ridolfi R,Ritter G, Rivoltini L, Romero PJ, Salem ML,Scheper RJ, Seliger BS, Sharma P, Shiku H,Singh-Jasuja H, Song W, Straten PT, TaharaH, Tian Z, van Der Burg SH, von Hoegen P,Wang E, Welters MJP, Winter H, WithingtonT, Wolchok JD, Xiao W, Zitvogel L, Zwi-erzina H, Marincola FM, Gajewski TF, Wig-ginton JM, Disis ML. Defining the criticalhurdles in cancer immunotherapy. J TranslMed, 9: 214, 2011.

8. Suzuki H, Takatsuka S, Akashi H, Yama-moto E, Nojima M, Maruyama R, Kai M,Yamano HO, Sasaki Y, Tokino T, ShinomuraY, Imai K, Toyota M. Genome-wide profil-ing of chromatin signatures reveals epige-netic regulation of microRNA genes in col-

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orectal cancer. Cancer Res, 71(17): 5646-5658,2011.

9. Ii M, Yamamoto H, Taniguchi H, Adachi Y,Nakazawa M, Ohashi H, Tanuma T, SukawaY, Suzuki H, Sasaki S, Imai K, Shinomura Y.Co-expression of laminin β3 and γ2 chainsand epigenetic inactivation of laminin α3chain in gastric cancer. Int J Oncol, 39(3):593-599, 2011.

10. Taniguchi H, Jacinto F.V., Villanueva A, Fer-nández AF, Yamamoto H, Carmona F.J,Puertas S, Marquez VE, Shinomura Y, ImaiK, and Esteller M. Silencing of the Kruppel-like factor 2 by the histone methyltransferaseEZH2 in human cancer. Oncogene, AdvanceOnline Publication, 2011, doi: 10.1038/onc.2011.387.

11. Ii M, Li H, Adachi Y, Yamamoto H, OhashiH, Arimura Y, Carbone DP, Imai K, Shi-nomura Y. The efficacy of IGF-I receptormonoclonal antibody against human gastro-intestinal carcinomas is independent of k-rasmutation status. Clin Cancer Res, 17(15):5048-5059, 2011.

12. Tanaka H, Arimura Y, Yabana T, Goto A,Hosokawa M, Nagaishi K, Yamashita K,Yamamoto H, Sasaki Y, Fujimiya M, Imai K,Shinomura Y. Myogenic lineage differenti-ated mesenchymal stem cells enhance recov-ery from dextran sulfate sodium-induced co-litis in the rat. J Gastroenterol, 46: 143-152,2011.

13. Kamimae S, Yamamoto E, Yamano HO, No-jima M, Suzuki H, Ashida M, Hatahira T,Sato A, Kimura T, Yoshikawa K, Harada T,Hayashi S, Takamaru H, Maruyama R, KaiM, Nishiwaki M, Sugai T, Sasaki Y, TokinoT, Shinomura Y, Imai K, Toyota M. Epige-netic alteration of DNA in mucosal washfluid predicts invasiveness of colorectal tu-mors. Cancer Prev Res, 4(5): 674-683, 2011.

14. Sasaki S, Ishida T, Toyota M, Ota A, SuzukiH, Takaoka A, Yasui H, Yamamoto H, Taka-gi H, Maeda M, Seito T, Tsujisaki M, Shino-mura Y, Imai K. Interferon-α/β and Anti-Fibroblast Growth Factor Receptor 1 Mono-clonal Antibody Suppress Hepatic CancerCells in Vitro and in Vivo. PLoS ONE, 6(5):e19618, 2011.

Project Professor, Kouhei Tsumoto:1. Satoshi Ohtake, Kouhei tsumoto, Masao

Tokunaga, Yoshiko Kita, Tsutomu Arakawa.The mechanism of elution by MgCl2, ethyl-ene glycol and arginine in affinity chroma-tography. Global Journal of Biochemistry, 2:1-27, 2011.

2. Yoshitaka Moriwaki, Jose M.M. Caaveiro,Yoshikazu Tanaka, Hiroshi Tsutsumi, ItaruHamachi, Kouhei Tsumoto. Molecular Basis

of Recognition of Antibacterial Porphyrinesby Heme-Transfer IsdH-NEAT3 of Staphylo-coccus aureus. Biochemistry, 50: 7311-7320,2011.

3. Nagatoishi S, Isono N, Tsumoto K, Sugi-moto N. Hydration is Required in DNA G-Quadruplex-Protein Binding . ChemBio-Chem, 12: 1822-1826, 2011.

4. Suzuki T, Kim M, Kozuka-Hata H, Wata-nabe M, Oyama M, Tsumoto K, YamamotoT. Monoubiquitination of Tob/BTG familyproteins competes with degradation-targeting polyubiquitination. Biochemicaland Biophysical Research Communications,409: 70-74, 2011.

5. Yachi K, Ohta T, Fukushima T, Watanabe T,Katayama Y, Okamoto Y, Sano E, TsumotoK. Gene expression profiles predicting theresponse to IFN-β and a combination of te-mozolomide and IFN-β in malignantgliomas. International Journal of Oncology,39: 529-542. 2011.

6. Satoru Nagatoishi, Noburu Isono, KouheiTsumoto and Naoki Sugimoto. Contributionof loops in DNA G-quadruplex on the bind-ing of protein. Biochimie, 93: 1231-1238,2011.

7. Ishii D, Abe R, Watanabe SI, Tsuchiya M,Nöcker B, Tsumoto K. Stepwise Characteri-zation of the Thermodynamics of TrichocyteIntermediate Filament Protein Supramolecu-lar Assembly. J. Mol. Biol., 408: 832-838,2011.

8. Rahul S. Rajan, Kouhei Tsumoto, MasaoTokunaga, Hiroko Tokunaga, Yoshiko Kitaand Tsutomu Arakawa. Chemical and Phar-macological Chaperones: Application for Re-combinant Protein Production and ProteinFolding Diseases. Current Medicinal Chem-istry, 18, 1-15, 2011.

9. Yanaka S, Sano E, Naruse N, Miura KI,Futatsumori-Sugai M, Caaveiro JM, TsumotoK. Non-core region modulates interleukin-11signaling activity: Generation of agonist andantagonist variants. The Journal of BiologicalChemistry, 286: 8085-8093, 2011.

10. Motonori Kudou, Ryosuke Yumioka, Daisu-ke Ejima, Tsutomu Arakawa, Kouhei Tsu-moto. A novel protein refolding system us-ing lauroyl-l-glutamate as a solubilizing de-tergent and arginine as a folding assistingagent. Protein Expression and Purification,75: 46-54, 2011.

11. Motonori Kudou, Daisuke Ejima, HarunaSato, Ryosuke Yumioka, Tsutomu Arakawa,Kouhei Tsumoto. Refolding single-chain an-tibody (scFv) using lauroyl-L-glutamate as asolubilization detergent and arginine as a re-folding additive. Protein Expression and Pu-

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rification, 77: 68-74, 2011.12. Mihoko Ui, Yoshikazu Tanaka, Takeshi

Tsumuraya, Ikuo Fujii, Masayuki Inoue,Masahiro Hirama, and Kouhei Tsumoto.Structural and Energetic Hot-Spots for theInteraction between a Ladder-like PolycyclicEther and the Anti-Ciguatoxin Antibody 10C9Fab. Molecular BioSystems, 7: 793-798,2011.

Project Assistant Professor , TaniguchiHiroaki:1. Ohashi H, Adachi Y, Yamamoto H, Tani-

guchi H, Nosho K, Suzuki H, Arimura Y,Imai K, Carbone DP, Shinomura Y. Insulin-like growth factor receptor expression is as-sociated with aggressive phenotypes andhas therapeutic activity in biliary tract can-cers. Cancer Sci. 2011 Nov 2. Epub ahead ofprint.

2. Taniguchi H, Jacinto F.V., Villanueva A, Fer-nández AF, Yamamoto H, Carmona F.J,Puertas S, Marquez VE, Shinomura Y, ImaiK, and Esteller M. Silencing of the Kruppel-like factor 2 by the histone methyltransferaseEZH2 in human cancer. Oncogene, AdvanceOnline Publication, 2011, doi: 10.1038/onc.2011.387.

3. Ii M, Li H, Adachi Y, Yamamoto H, OhashiH, Taniguchi H, Arimura Y, Carbone DP,Imai K, Shinomura Y. The Efficacy of IGF-IReceptor Monoclonal Antibody against Hu-

man Gastrointestinal Carcinomas is Inde-pendent of k-ras Mutation Status. Clin Can-cer Res, 17: 5048-5059. 2011.

4. Ii M, Yamamoto H, Taniguchi H, Adachi Y,Nakazawa M, Ohashi H, Tanuma T, SukawaY, Suzuki H, Sasaki S, Imai K, Shinomura YCo-expression of laminin β3 and γ2 chainsand epigenetic inactivation of laminin α3chain in gastric cancer. Int J Oncol, 39: 593-599. 2011.

5. Fernandez AF, Assenov Y, Martin-Subero JI,Balint B, Siebert R, Taniguchi H, YamamotoH, Hidalgo M, Tan AC, Galm O, Ferrer I,Sanchez-Cespedes M, Villanueva A, Car-mona J, Sanchez-Mut JV, Berdasco M,Moreno V, Capella G, Monk D, Ballestar E,Ropero S, Martinez R, Sanchez-Carbayo M,Prosper F, Agirre X, Fraga MF, Graña O,Perez-Jurado L, Mora J, Puig S, Prat J, Badi-mon L, Puca AA, Meltzer SJ, Lengauer T,Bridgewater J, Bock C, Esteller M. A DNAmethylation fingerprint of 1628 human sam-ples. Genome Res. 2011 Jul 12. Epub aheadof print.

6. Hizaki K, Yamamoto H, Taniguchi H, Ada-chi Y, Nakazawa M, Tanuma T, Kato N,Sukawa Y, Sanchez JV, Suzuki H, Sasaki S,Imai K, Shinomura Y. Epigenetic inactiva-tion of calcium-sensing receptor in colorectalcarcinogenesis. Mod Pathol, 24: 876-884,2011.

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The aim of our division is to establish and popularize state-of-art medicine and topromote translational research (TR). We investigate a methodology to develop na-tional consensus in health care by using media.We also perform individual case studies on H1N1 pandemic, economic burden ofhealth care on patients, medical support for disaster-stricken area by the GreatEast Japan Earthquake on March 11, 2011 and clinical internship system. In eachcase, we also study the system of management, information circulation, and net-work.

【Medical Governance】

Tomoko Matsumura, Naoko Murashige, YukoKodama, Haruka Nakada, Koichiro Yuji1,Masaharu Tsubokura, Masahiro Kami: 1De-partment of Medicine (Department of Hematol-ogy/Oncology), Institute of Medical Science,The University of Tokyo.

We perform individual case studies on H1N1pandemic, economic burden of health care onpatients, and clinical internship system. In addi-tion, we also study the system of management,information circulation, and network in eachcase and publish the results in scientific journals(Ohara M, et al., Yuji K, et al., Murashige N, etal. Yuji K, et al.). These results are widely pub-lished in news papers and popular magazines.

【Medical support for disaster-stricken area】

Masaharu Tsubokura, Yukio Kanazawa2 ,Tomoyoshi Oikawa2, Kyohei Takahahi3, AkemiTakada2 , Tomoko Matsumura , Morihito

Takita, Tamae Hamaki4, Kazuhiko Kobayashi5,Syuichi Iwamoto4, Jinichi Mori6, Yukie Taka-hashi6, Masaki Miyasaka4, Hideki Komatsu7,Makoto Suzuki7, Mamiko Ohara7, TsunehikoKomatsu8, Tetsuya Miyashita9, Tetsuya Tani-moto10, Yuko Kodama, Masahiro Kami:2Minamisoma Municipal General Hospital,3Haramachi Central Maternity Clinic, 4TokyoMetropolitan Bokutoh Hospital, 5JR TokyoGeneral Hospital, 6Tokyo Metropolitan Cancerand Infectious diseases Center Komagome Hos-pital, 7Kameda Medical Center, 8YokohamaCity University Hospital, 9Teikyo UniversityChiba Medical Center, 10Pharmaceuticals andMedical Devices Agency, JAPAN.

Collaborating with physicians who work indisaster-stricken area and many support physi-cians, we conducted rescue and evacuation ac-tivities for patients with hemodialysis and pub-lish the results in scientific journals (TsubokuraM, et al.).

Donatinon Laboratories

Division of Social Communication Systemfor Advanced Clinical Research先端医療社会コミュニケーションシステム社会連携研究部門

Project Professor Masahiro Kami, M.D., Ph.D.Project Assistant Professor Tomoko Matsumura, M.D., Ph.D.

特任教授 医学博士 上 昌 広特任助教 医学博士 松 村 有 子

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【Information Circulation on the AdvancedMedicine】

Yukiko Sakamoto-Kishi, Naoko Murashige,Masahiro Kami, Tomoko Matsumura,

We investigate the role of the manga in thehealth care (Kishi Y, et al.). Through internetsearches using Google, we investigated the char-acteristics of medical manga published in Japan,defined as those in which the main character isa medical professional and that occur in a medi-cal setting. In our survey, it was suggested thatMedical manga would be a powerful tool for in-creasing the awareness of the public regardingmedicine.

【Economic Burden of Health Care on Pa-tients】

Yuko Kodama, Ryoko Morozumi7, AkihikoMatsui8, Masahiro Kami, Tomoko Matsumura:7Faculty of Economics, University of Toyama,Toyama, Japan, 8Faculty of Economics, theUniversity of Tokyo, Tokyo, Japan.

Imatinib (Glivec), which is the first-line drugfor chronic myelogenous leukemia (CML), ishighly efficient. We clarified that the cost ofGlivec in Japan was higher than the other coun-tries by international research. We conduct acollaborate study with Professor Matsui at Fac-ulty of Economics, the University of Tokyo, toreview the utilization of Glivec and its cost inJapan. Because the economic burden on patientsor the government with prevailing advancedmedical care including anticancer drugs is animportant issue, we continue further investiga-tion.

Publications

1. Ohara M, Tsubokura M, Naoto H, Kami M,Mochizuki T. H1N1 influenza A outbreakamong young medical staff members whoreceived single dose of non-adjuvanted split-virion 2009 H1N1 vaccine. Hum Vaccin. 7:56-57, 2011.

2. Yuji K, Oiso G, Matsumura T, Murashige N,Kami M. Police investigation into multidrug-resistant acinetobacter baumannii outbreakin Japan. Clin Infect Dis. 52: 422, 2011.

3. Murashige N, Nonaka S, Kodama Y, Matsu-mura T, Kami M. Lessons from Japanesephysicians’ education and UK budget in-crease. Lancet. 377: 1239, 2011.

4. Yuji K, Narimatsu H, Tanimoto T, KomatsuT, Kami M. Sharing information on adverseevents. Lancet. 377: 1654, 2011.

5. Tsubokura M, Horie S, Komatsu H, TokiwaM, Kami M. The impact of the Great To-hoku Earthquake on the dialysis practice inthe disaster-stricken area. Hemodial Int.2011.

6. Kishi Y, Matsumura T, Murishige N, Ko-dama Y, Hatanaka N, Takita M, et al.Internet-based survey on medical manga inJapan. Health Commun. 26: 676-8, 2011.

7. Kodama Y, Matsumura T, Yamaguchi T,Takita M, Kawagoe S, Kimura Y, et al. Age,gender, will, and use of home-visit nursingcare are critical factors in home care for ma-lignant diseases; a retrospective study in-volving 346 patients in Japan. BMC PalliatCare. 10: 17, 2011.

8. Mori J, Tsubokura M, Kami M. Cytarabinedose for acute myeloid leukemia. N Engl JMed. 364: 2166-2167; author reply 8-9, 2011.

9. Narimatsu H, Kakinuma A, Sawa T,Komatsu T, Matsumura T, Kami M, et al.Usefulness of a bidirectional e-learning ma-terial for explaining surgical anesthesia tocancer patients. Ann Oncol. 22: 2121-2128,2011.

10. Takahashi Y, Mori J, Kami M. BRAF muta-tions in hairy-cell leukemia. N Engl J Med.365: 960-962; author reply 1-2, 2011.

11. Takahashi Y, Mori J, Tsubokura M, Matsu-mura T, Kami M. Hydroxycarbamide use inyoung children with sickle-cell anaemia.Lancet. 378: 1777, 2011.

12. Takita M, Tanaka Y, Kodama Y, MurashigeN, Hatanaka N, Kishi Y, et al. Data miningof mental health issues of non-bone marrowdonor siblings. J Clin Bioinforma. 1: 19,2011.

13. Tanimoto T, Sakiyama M, Hori A, YagasakiF, Kami M. Rituximab-containing therapyfor chronic lymphocytic leukaemia. Lancet.377: 205; author reply 6, 2011.

14. Tanimoto T, Matayoshi T, Yagasaki F,Takeuchi K, Kami M. Safety and efficacy ofzoledronic acid in multiple myeloma. Lan-cet. 377: 2178, 2011.

15. Tsubokura M, Miura Y, Itokawa T, MurataK, Takei N, Higaki T, et al. Fatal dysrhyth-mia following initiation of lansoprazole dur-ing a long-term course of voriconazole. JClin Pharmacol. 51: 1488-1490, 2011.

16. Tsubokura M, Yamashita T, Inagaki L, Ko-bayashi T, Kakihana K, Wakabayashi S, etal. Fatal intracranial hemorrhage followingadministration of recombinant thrombo-modulin in a patient after cord blood trans-

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plantation. Bone Marrow Transplant. 46:1030-1031, 2011.

17. Watanabe N, Noh JY, Narimatsu H,Takeuchi K, Yamaguchi T, Kameyama K, etal. Clinicopathological features of 171 casesof primary thyroid lymphoma: a long-termstudy involving 24-553 patients with Hashi-moto’s disease. Br J Haematol. 2011.

18. Watanabe N, Narimatsu H, Noh JY, Yama-guchi T, Kobayashi K, Kami M, et al. An-tithyroid drug-induced hematopoietic dam-age: a retrospective cohort study of agranu-locytosis and pancytopenia involving 50,385patients with Graves’ disease. J Clin Endo-crinol Metab. 97: E49-53,2012.

19. Nazgul A. Omurzakova, Yoshihisa Yamano,Tomoo Sato, Toshihiko Izumi, KazukoAzakami, Daisuke Hasegawa, Ryoji Fujii,Naoko Yagishita, Satoko Aratani, Zuhra S.Kabaeva, Mirsaid M. Mirrakhimov, Masa-

hiro Kami, Ikuro Maruyama, MitsuhiroOsame, Shunpei Yokota, Kusuki Nishioka,Toshihiro Nakajima. Increased prevalence ofgroup A β-hemolytic streptococcus amongan ethnic population in Kyrgyzstan detectedby the rapid antigen detection test. Molecu-lar Medicine Report. 4: 869-874, 2011.

20. Hatanaka N, Takita M, Yamaguchi T, KamiM, Matsumoto S. Development of a novelscale to assess the quality of life in type 1diabetic patients for beta cell replacementtherapy. Diabetology International. 2: 55-64;2011.

21. Murashige N, Matsumura T, Masahiro K.Disseminating Japan’s immunisation policyto the world. Lancet. 377: 299, 2011.

22. Murashige N. Japan’s immunisation policyin routine, pandemic and post-tsunami situ-ations. Int J Clin Pract. 65: 1126-1131, 2011.

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In the division of “Interactome Medical Sciences”, our developed technology in Ja-pan, called as “puromycin technology”, is used as analysis tools of interactome (i.e. comprehensive biomolecular interactions). Toward order-made medicine era, wewill apply these tools to medical science. Interdisciplinary approach is required toaccomplish our research. To develop the “interactome analysis pipeline”, we col-laborate with human genome center, where large sequence data is yielded fromnext generation sequencer and analyzed with supercomputer. Especially, we focuson “cancer” because of its diversity and individuality, and cancer researchers arealso collaborated in our research. For cooperation study with the society and aca-demia, industries are involved in our research.

Understanding and Control of Cancer StemCells for Realizing Personalized GenomicMedicine

Shigeo Fujimori1, Takatsune Smimizu2, SeiyaImoto3, Masao Nagasaki3, Rui Yamaguchi3,Naoya Hirai1, Kazuyo Masuoka1, TaisukeMori4, Hideyuki Saya2, Satoru Miyano3, EtsukoMiyamoto-Sato1,3: 1Div. Interactome Med. Sci.,Inst. Med. Sci., Univ. Tokyo, 2Div. Gene Reg.,IAMR, Keio Univ. Sch. Med, 3Human GenomeCtr., Inst. Med. Sci., Univ. Tokyo, 4Mol. Pathol.Div., Natl. Cancer. Ctr. Res. Inst.

On the verge of gene therapy, the interest ofbasic research in the medical care has transitedfrom the conventional post-genomic to the per-sonal genomic research. Especially, cancer is oneof the diseases requiring personal genome medi-cine. To understand the individuality of cancer,we have to conduct various sorts of omicsanalyses and accurately comprehend the indi-vidual state of complicated biomolecular interac-tion networks by cell types. Our research projectfocuses on diversity of cancer stem cells (CSCs),and is aimed at discerning and regulation of

CSCs by the collaboration of researchers havingthe model experiment system for studyingCSCs, having technology for the omics dataanalysis and having network analysis techniquebases on mathematical models. Different typesof CSC model cells (Shimizu et al., Oncogene.2010) are used in our research. Those CSCmodel cells that are derived from bone-marrowstromal cells of Ink4a KO mice and can be in-duced in vitro by the over expression of c-Myc.Although all of those CSCs cause osteosarcomato develop after graft to the mouse, differ in ma-lignancy. In order to compare those CSCs hav-ing different characteristics using omics dataand systems biology techniques, we plan to con-duct an analysis of large scale biomolecular in-teraction (interactome) in different CSC modelsusing the in vitro virus method (IVV method;Miyamoto-Sato E. et al., PLoS ONE. 2010) cou-pled with next generation sequencer. The IVVmethod is our originally developed technologyfor protein analysis and is able to determine notonly ‘interacting partners’ of the protein, butalso ‘interacting region (domain)’ simultane-ously, allowing us to analyze relationship be-tween interactome and aberrance (e.g., splicing

Donatinon Laboratories

Division of Interactome Medical Sciencesインタラクトーム医科学社会連携研究部門

Project Associate Professor Etsuko Miyamoto-Sato, Ph.D. 特任准教授 工学博士 宮 本 悦 子

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abnormality and DNA mutation), which are fre-quently observed in cancer cells. Further, wehave developed the database to investigate suchrelationship between them.

Highly efficient analysis of interactome usingthe in vitro virus method combined withhigh-throughput sequencing

Shigeo Fujimori1, Naoya Hirai1, KazuyoMasuoka1 , Takanori Washio1,2 , TomohiroOshikubo1,3, Tatsuhiro Yamashita1,4, EtsukoMiyamoto-Sato1,5: 1Div. Interactome Med. Sci.,Inst. Med. Sci., Univ. Tokyo, 2RIKEN GENE-SIS Co., Ltd., 3Fujitsu Adv. Eng. Ltd., 4BioITBiz. Dev. Unit, Fujitsu Ltd., 5Human GenomeCtr., Inst. Med. Sci., Univ. Tokyo.

Current high-throughput ‘interactome’ da-tasets have not only low coverage but also lowreliability yet. Although next generation se-quencing (NGS) has been applied in various re-searches of molecular biology, it is not easy toutilize for analyzing interactome. In this re-search, to address these issues, we developed anIVV-HiTSeq method as an in vitro virus (IVV)method combined with NGS for the interactomeanalysis. The IVV method is one of the mRNAdisplay methods and has often been utilized forthe selection of prey proteins with bait from thelarge cDNA library. This technology is based onthe technique of the formation of linkage be-tween mRNA as genotype and their coding pro-tein as phenotype, thereby an iteration of selec-tion and amplification processes using mRNAtags for the enrichment is realized. Prey mole-cules enriched through iteration of severalrounds of selection can easily be detected by se-quencing of RNA moiety. In the IVV-HiTSeqmethod, prey DNA samples amplified with bar-coded primers representing the round of selec-tion are sequenced simultaneously. Obtainedread data can be used as substitute for quantita-tive real-time PCR assays and has an ability toevaluate the statistical significance of interac-tions. We confirmed that our method can im-prove both the coverage and reliability of inter-actome datasets. Furthermore, our method has apotential to be applied to not only in vitro selec-tions of PPI but also to detections of proteinsthat interact with nucleic acids or chemicals,suggesting that our method should be useful forexploring large space of protein sequences.

IRView: a database and viewer for protein in-teracting regions

Shigeo Fujimori1, Naoya Hirai1, KazuyoMasuoka1, Tomohiro Oshikubo1,2, Tatsuhiro

Yamashita1,3 , Takanori Washio1,4 , AyumuSaito5, Masao Nagasaki5, Satoru Miyano5,Etsuko Miyamoto-Sato1,5 : 1Div. InteractomeMed. Sci., Inst. Med. Sci., Univ. Tokyo, 2Fu-jitsu Adv. Eng. Ltd., 3BioIT Biz. Dev. Unit, Fu-jitsu Ltd., 4RIKEN GENESIS Co., Ltd., 5Hu-man Genome Ctr., Inst. Med. Sci., Univ. To-kyo.

Protein-protein interactions (PPIs) are medi-ated through specific regions (sites/domains) onproteins. Some proteins have two or more pro-tein interacting regions (IRs) and some of thoseIRs are competitively used for interactions withdifferent proteins. To facilitate comprehension ofsuch complicated interaction patterns at the re-gion level, we developed the IRView, an onlinedatabase that allows viewing of IRs in proteinsequences. Currently, the IRView contains 3,417IRs of human and mouse proteins obtainedfrom several different sources, together withother annotated region data, such as InterProdomain/motif regions, non-synonymous singlenucleotide polymorphism (nsSNP) sites andvariable regions owing to alternative mRNAsplicing. Since all region data in the IRView arestored and displayed as standardized positionson reference sequences, users can intuitivelycomprehend positional relations of each regionto others via the user-friendly web interface. Allrecords can be easily browsed and queried tosearch proteins of interest. The content of thedatabase will be updated regularly. The IRViewis publicly available at http://ir.hgc.jp/

PRD: A protein-RNA interaction database

Shigeo Fujimori1, Katsuya Hino2, AyumuSaito3, Masao Nagasaki3, Satoru Miyano3,Etsuko Miyamoto-Sato1,2,3: 1Div. InteractomeMed. Sci., Inst. Med. Sci., Univ. Tokyo, 2Dept.Biosci. & Info., Keio Univ., 3Human GenomeCtr., Inst. Med. Sci., Univ. Tokyo.

Although protein-RNA interactions (PRIs)have essential roles in a variety of cellular proc-esses, compiled data on PRIs at the gene levelare scarce compared with protein-protein inter-actions, which have been intensively surveyedand accumulated in public databases. Here, wepresent a PRI database (PRD) that provides in-formation on PRIs described in the scientific lit-erature . Currently the database contains＞10,000 interactions imported from public data-bases and ＞400 originally curated interactions.Each interaction is linked to genes, articles, andtaxonomy names, with identifiers used in theNCBI database. Furthermore, each record con-tains detailed information regarding protein

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binding regions, conserved RNA elements, anddetection methods (when available). Interactiondata in our database were curated and storedaccording to the PSI-MI standard. Users canbrowse all recorded interactions and executeflexible keyword searches against the database

via a web interface. Our database is not only areference of PRIs, but will also be a valuable re-source for studying characteristics of PRI net-works. PRD can be freely accessed at http://pri.hgc.jp/. The content of the database will be con-tinually updated.

Publications

Etsuko Miyamoto, Toru Tsuji, Shigeo Fujimori,Masamichi Ishizaka: CONSTITUTION OFTOOL FOR ANALYZING BIOMOLECULARINTERACTION AND ANALYSIS METHODUSING SAME. PCT/JP2011/058203 (2011)宮本悦子、堀澤健一、柳川弘志：c-Junタンパク質と複合体を形成するタンパク質、及び，それをコードする核酸、ならびに、それらの利用方法．特願２００５―５１６４３２（名義変更日：平成２３年９月１４日）

Etsuko Miyamoto, Kenichi Horisawa, HiroshiYanagawa: PROTEIN FORMING COMPLEXWITH c-Jun PROTEIN, NUCLEIC ACID EN-CODING THE SAME AND METHOD OF US-ING THE SAME. US Patent 7838243 (29th,Nov. 2011)

Etsuko Miyamoto, Kenichi Horisawa, HiroshiYanagawa: PROTEIN FORMING COMPLEXWITH c-Jun PROTEIN, NUCLEIC ACID EN-CODING THE SAME AND METHOD OF US-ING THE SAME. German Patent Application

No. 11 2004 002 217.7-41 (29th, Nov. 2011)宮本悦子，藤森茂雄：標的物質と相互作用するタンパク質の検出方法．国立大学法人東京大学，特願２０１１―２１３４２４（２０１１）宮本悦子，柳川弘志：翻訳テンプレートおよびそのライブラリー，それから合成される蛋白質および蛋白質のライブラリー、ならびにそれらを構成する要素、ならびにそれらの製造方法および利用方法，学校法人慶應義塾、特許２００４―５１３４８８；登録４７４７２９２（２０１１）宮本悦子：日の丸印のテクノロジーの誕生ーポストゲノム時代からパーソナル時代へ向けて．化学経済５月号：７３―７６（２０１１）藤森茂雄、森泰昌、佐谷秀行、宮本悦子：タンパク質間相互作用ネットワーク解析によるがん個性の理解．システムがんNewsletter ２：４（２０１１）

Hiroyuki Ohashi, Etsuko Miyamoto-Sato: A nextgeneration evolutionary molecular engineer-ing system and the future prospects. Vivaorigino 40 suppl (2012)

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Our major research interest is to elucidate the molecular mechanisms regulatingcancer cells, stem cells, cancer stem cells and development. Our team has twoimportant research directions: One is to clarify the basic principles underlying biol-ogy and the other is to apply the knowledge extracted from the basic principles totranslational medicine. In particular, we are focusing on growth factor signaling,such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). In or-der to achieve the goal, we take a challenging combinatorial approaches of mo-lecular biology and systems biology, in addition to conventional methods of mo-lecular biology.

1. Translational medicine by using systemsbiology approach

Asuka Nakata, Anna Mizutani, Tasunori Nishi-mura, Rui Yamaguchi1, Masao Nagasaki1, Tep-pei Shimamura1, Seiya Imoto1, Ayumu Saito1,Ryo Yoshida2, Tomoyuki Higuchi2, TakashiKohno3, Jun Yokota3, Satoru Miyano1, NorikoGotoh1: 1Human Genome Center, Institute ofMedical Science, University of Tokyo, Tokyo,Japan, 2Institute of Statistical Mathematics, To-kyo, Japan, 3Biology Division, National CancerCenter Research Institute.

1-1. EGF receptor tyrosine kinase definescritical prognostic genes of stage IAlung adenocarcinoma

Non-small cell lung cancer (NSCLC) is thecommonest and the most fatal histological sub-type of lung cancer. About 10-30％ of stage IApatients die due to recurrence after surgery of

curative intent. Therefore, the identification ofprognostic biomarkers for stage IA lung adeno-carcinoma with poor prognosis is of great im-portance to select patients who will be benefitedby adjuvant therapy. To the best of our knowl-edge, there is little evidence that gene signaturesare useful above the clinical prognostic markersalone, including stage, age and gender. Epider-mal growth factor (EGF) signaling pathway isclosely related to aggressive phenotypes of lungand other cancers. EGF receptor (EGFR) tyrosinekinase-specific inhibitor, namely gefitinib, is ex-pected to alter the gene expression patternscaused by EGFR tyrosine kinase activity. Weperformed DNA microarray analysis in order toobtain the comprehensive gene expression pro-files in human primary lung epithelial cells thatwere stimulated with EGF in the presence or ab-sence of gefitinib. The detailed time courses arecomposed of 19 time points along 48 hours. Thedata were subjected to a unique mathematicalanalysis by using the State Space Model (SSM)

Collaborative Research Unit

Division of Systems Biomedical Technology(Konika Minolta Technology Center)システム生命医科学技術開発共同研究ユニット（コニカミノルタテクノロジーセンター）

Project Associate Professor Noriko Gotoh, M.D., Ph.D.Project Assistant Professor Kunihiko Hinohara, Ph.D.

特任准教授 医学博士 後 藤 典 子特任助教 医学博士 日野原 邦 彦

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in order to select genes of which expression pat-terns were altered by gefitinib. We indentifiedsuch 139 genes as the EGFR tyrosine kinase-influenced key genes. The 139 EGFR tyrosinekinase-influenced key genes were used as ex-pression signatures to train a risk scoring modelthat classifies patients in high- or low-risk (therisk of death or recurrence in 5 years). Thismodel was trained by using a data set com-posed of 253 North American patients with lungadenocarcinomas. Then, the predictive ability ofthe risk scoring model was examined in two in-dependent cohorts composed of North Ameri-can and Japanese patients. The model enabledthe statistically significant identification of high-risk stage IA lung adenocarcinoma in both co-horts, with hazard ratios (HRs) for death of 7.16(P＝0.029) for North American and of 10.98 (P＝0.008) for Japanese.

The set of 139 genes includes many ones thathave already been reported in the literature tobe involved in biological aspects of cancer phe-notypes such as ADAM family genes (ADAM10,ADAM19), matrix metalloprotease (MMP) fam-ily genes, NMU and Ube2c, but are yet un-known to be involved in EGF signaling. Theseresults strongly re-emphasizes that EGF signal-ing status underlies aggressive phenotype ofcancer cells, and also suggests the first set ofgenes that are useful for the identification ofhigh-risk stage IA lung adenocarcinoma pa-tients.

Furthermore, we found that expression levelsof a single gene of 72/139 genes are signifi-cantly associated with the survival and/orrelapse-free survival of the adenocarcinoma pa-tients when stage IA, IB and II patients are com-bined. Among the 72 genes, expression levels ofa single gene of 40 genes are significantly associ-ated with poor prognosis. We are now examin-ing the possibility that there are novel moleculartargets for cancer therapy among them.

1-2. Novel molecular mechanisms of ac-quired resistance to gefitinib in Non-small lung cancer

EGFR tyrosine kinase inhibitors (EGFR-TKIs),such as gefitinib or erlotinib, were remarkablyeffective in some patients with NSCLC harbor-ing EGFR kinase domain mutation. Althoughsuch NSCLC patients were initially sensitive toEGFR-TKIs, they eventually have acquired resis-tance to EGFR-TKIs. There are several molecularmechanisms of acquired resistance to EGFR-TKI;a secondary mutation in EGFR(T790M) and theamplification of the MET oncogene are com-monly found. However, some NSCLC patientsstill suffer from gefitinib-resistance caused by

unknown mechanisms.To explore novel molecular mechanisms for

gefitinib-resistance, we established the gefitinib-resistant PC9M2 cells that were spontaneouslyderived from PC9 cells expressing gefitinib-sensitive mutant EGFR, by culturing them in thepresence of a low amount of gefitinib for severalmonths. In PC9M2 cells, we found neither themutation of EGFR T790M nor amplification ofMET oncogene and thus the novel mechanismsmay underlie the acquired resistance to gefitinibin these cells.

We analyzed gene expression profiles of PC9and PC9M2 cells in a detailed time course withor without stimulation with EGF in the presenceor absence of gefitinib by using DNA microar-ray. Bioinformatics analysis revealed that ex-pression levels of several genes in Wnt signalingpathways were increased in PC9M2 cells com-pared with those in PC9 cells. Furthermore, wefound the phosphorylation of GSK3 and the nu-clear accumulation of β-catenin were increasedin PC9M2 cells. Our approach would provide auseful strategy to find appropriate moleculartargets for cancer patients who are sufferingfrom acquired resistance to cancer drugs.

1-3. Search for new lung cancer biomarkersand molecular targets

Although patients with lung cancer at theearly stages may be curable by surgery, theyhardly recognize to have the disease, becausethere is no symptom. It is thus important to de-tect lung cancer patients at early stages by ex-amining serum biomarkers. However, useful se-rum biomarkers that are able to detect lung can-cer patients at early stages remain to be identi-fied.

In this study, we searched for new serumbiomarkers for the early stage lung cancer byanalyzing expression profiles of stage I lungadenocarcinoma tissues derived from ～200patients who underwent surgery in NationalCancer Center Hospital. As a result, 24 candi-date molecules were obtained. We collectedplasma of all these patients and their plasmasamples are subject to enzyme-linked immuno-sorbent assay (ELISA) to measure expressionlevels in plasma in order to validate them ascandidates.

The protein levels of 23 candidates were ex-amined by ELISA using 10 plasma samples from2 healthy people and 7 stage I lung adenocarci-noma patients at the first screening. We selected6 candidate molecules for the second screening.The candidate molecules are CXCL13, ADAM8,COL10A1, FGL1, GPR37 and MUC5B. We used53 plasma samples from 9 healthy people and

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44 stage I lung adenocarcinoma patients. Afterthe second screening, fibrinogen-like 1 (FGL1)turned out to be a strong candidate for the earlydetection biomarker (AUC＝0.684). According toRefExA database of LSBM, FGL1 mRNA is ex-pressed in normal liver, normal pancreas, fetalliver, a few liver cancer cell lines and some lungcancer cell lines.

2. Breast cancer stem cells

2-1. ErbB/NF-κB signaling regulates breastcancer stem cell-like properties

Kunihiko Hinohara, Seiichiro Kobayashi4, Ari-nobu Tojo4, Jun-ichiro Inoue5, Noriko Gotoh:4Division of Molecular Therapy, 5Division ofCellular and Molecular Research.

Cancer stem cells, a small proportion of het-erogeneous tumor cells, can self-renew and si-multaneously produce differentiated tumor cellswith strong proliferative activity, and thereforeare responsible for tumorigenesis. Although it isimportant to target cancer stem cells for treat-ment of cancer patients, it has been difficult tofind appropriate target molecules because themechanisms by which cancer stem cells main-tain their ability remain obscure. Here, we iden-tified a molecular mechanism that regulatesbreast cancer stem cell (BCSC)-like properties.We found that heregulin (HRG), a ligand forErbB3, induced mammosphere formation inbreast cancer stem cells (BCSCs) as well as inbreast cancer cell lines. HRG-induced mam-mosphere formation was reduced by treatmentwith inhibitors for phosphatidyl inositol 3-kinase (PI3K) or NF-κB and by expression ofIκBα-Super Repressor (IκBαSR), a dominant-negative inhibitor for NF-κB. Moreover, theoverexpression of IκBαSR in breast cancer cellsinhibited tumorigenesis in NOD/SCID mice.Furthermore, we found that the expression of IL8, a regulator of BCSC self-renewal, was in-duced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate thatHRG/ErbB3 signaling appears to maintainBCSC properties through a PI3K/NF-κB path-way in human breast cancer.

2-2. Identification of ErbB/NF-κB target genesin breast cancer cells

Kunihiko Hinohara, Teppei Shimamura1, RuiYamaguchi1, Masao Nagasaki1, Seiya Imoto1,Satoru Miyano1, Noriko Gotoh

We performed time series microarray analysesto investigate transcriptome dynamics during

ErbB/NF-κB activation in breast cancer cellsupon stimulation with HRG, and identified 69genes positively regulated by ErbB/NF-κB sig-naling. Importantly, treatment of MCF7 cellswith several molecules encoded by these genes,including IGF2, SDF1α, SDF1β and IL20, in-creased mammosphere formation in a dose-dependent manner. These observations suggestthat the genes upregulated by ErbB/NF-κB sig-naling were important for maintaining themammosphere-forming abilities of breast cancercells.

3. FRS2 family adaptor protein

A potential role of an adaptor protein FRS2β,a feedback inhibitor for ErbB, for mainte-nance of dormancy of cancer stem-like cells

Annna Mizutani, Daisuke Iejima, Noriko Go-toh

The adaptor protein FRS2 family comprisestwo members: FRS2α and FRS2β. Although FRS2α is a well-known central mediator for FGFsignaling, the role of FRS2β for its physiologicaland pathological significance are still largely un-known. We have previously shown that FRS2βconstitutively binds to ErbB family membersand inhibits ErbB signaling, resulting in inhibi-tion of cell proliferation. One mechanism is thatit serves as a feedback inhibitor by binding tothe activated ERK after stimulation with ErbBtyrosine kinase. Another mechanism is that itpromotes degradation of ErbB2, resulting in re-duced protein amounts of ErbB2. We alsoshowed that expression of FRS2β is restricted toseveral tissues including epithelial cells in breastand lung tissues.

Immunohistochemical analysis revealed thatexpression of FRS2β was at low levels in non-pregnant mammary gland tissues, while duringpregnancy and lactation, it was increased in re-stricted areas of luminal epithelial cells in themammary gland tissues. Expression levels ofErbB2 were also increased during pregnancyand lactation. We found that expression levelsof ErbB2 and phospho-histone H3 (pH3), a pro-liferation marker, were reduced in cells in whichexpression of FRS2β was up-regulated. This isconsistent with the fact that FRS2β downregu-lates protein levels of ErbB2, leading to inhibi-tion of cell proliferation. These results promptedus to investigate the role of FRS2βduring tu-morigenesis in breast tissues.

We generated mutant mice in which Frs2βwas disrupted by gene targeting. The Frs2βknockout mice showed no gross abnormalityand healthy and fertile. We crossed the Frs2β

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knockout mice with mouse mammary tumor vi-rus (MMTV)-ErbB2/Neu transgenic mice inwhich overexpression of ErbB2 driven byMMTV promoter in mammary tissues inducesbreast cancer. Consistent with the fact thatFRS2β serves as a feedback inhibitor for ErbBsignaling, we found earlier onset of appearanceof mammary tumors in MMTV-ErbB2(＋)/Frs2β(－/－) mice than in MMTV-ErbB2(＋)/Frs2β(＋/＋) mice. However, tumor growth inMMTV-ErbB2(＋ )/Frs2β (－/－ ) mice wasgreatly reduced and eventually, all the MMTV-ErbB2(＋)/Frs2β (＋/＋) mice died faster thanthe MMTV-ErbB2(＋)/Frs2β (－/－) mice.

Expression levels of ErbB2 were unchangedbetween cells in which expression levels ofFRS2β were up-regulated or not in MMTV-ErbB2(＋)/Frs2β (－/－) mice, probably due to forcedexpression of ErbB2. In contrast, expression lev-els of pH3 were still reduced in cells in whichexpression of FRS2β was up-regulated, in asso-ciation with reduced nuclear translocation ofERK, as we previously reported. This strongly

suggests that ErbB-ERK axis is still inhibited incells expressing FRS2β.

Since low levels of cell proliferative activity isa characteristic of tumor-initiating cells or stem/progenitor cells, we examined the possibilitythat FRS2β plays roles for the functions of main-tenance of such cells. We found that the EGF-induced mammosphere forming ability of breastcancer cells and normal breast cells were de-creased in MMTV-ErbB2(＋)/Frs2β (－/－) micecompared with those of MMTV-ErbB2(＋)/Frsβ(＋/＋) mice, suggesting that tumor-initiatingcells and normal progenitor cells in breast tis-sues are decreased in MMTV-ErbB2(＋)/Frs2β(－/－) mice.

It thus appears that inhibition of ErbB-ERKaxis is important for the maintenance of bothtumor-initiating cells and normal progenitorcells, partly by keeping reduced cell growth ac-tivity. Taken together, we propose that ErbB sig-naling controls tumor-initiating cells and mam-mosphere formation through multiple ways.

Publications

1. Hinorara, K., Kobayashi S., Kanauchi, H.,Shimizu, S., Nishioka, K., Tsuji, E., Tada, K.,Umezawa, K., Mori, M., Ogawa, T., Inoue, J.,Tojo, A. & Gotoh, N.: ErbB/NF-kB signalingcontrols mammosphere formation in humanbreast cancer. Proc. Natl. Acad. Sci., USA,April 5, on line publication 2012.

2. Hinohara, K. & Gotoh, N.: NF-kB pathwaysin breast cancer stem cells for tumorigenesis.In: Breast cancer, Bentham eBooks, Bentham Sci-ence, in press.

3. Okayama, H., Khono, T., Ishii, Y., Shimada,Y., Shiraishi, K., Iwakawa, R., Furuta, K.,Tsuta, K., Shibata, T., Yamamoto, S., Wata-nabe, S., Sakamoto, H., Kumamoto, K., Take-noshita, S., Gotoh, N., Mizuno, H., Sarai, A.,Kawano, S., Yamaguchi, R., Miyano, S. &Yokota, J.: Identification of genes up-regulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinoma. CancerRes., 72, 100-111, 2012.

4. Nomura, M., Fukuda, T, Fujii, K., Kawamura,T., Tojo, H., Kihara, M., Bando, Y., Gazdar, A.F., Tsuboi, M., Oshiro, H., Nagao, T., Ohira,T., Ikeda, N., Gotoh, N., Kato, H., Marko-Varga, G. & Nishimura, T.: Preferential ex-

pression of potential markers for cancer stemcells in large cell neuroendocrine carcinomaof the lung. Journal of Clinical Bioinformatics, 1,23, (3 September), 2011.

5. Yamauchi, M., Yoshino, I., Yamaguchi, R.,Shimamura, T., Nagasaki, M., Imoto, S.,Niida, A., Koizumi, F., Kohno, T., Yokota, J.,Miyano, S. & Gotoh, N.: N-cadherin expres-sion is a potential survival mechanism ofgefitinib-resistant lung cancer cells. Am. J.Cancer. Res., 1, 823-833, 2011.

6. Gotoh, N.: Somatic mutations of the EGF re-ceptor and their signal transducers affect theefficacy of EGF receptor-specific tyrosinekinase inhibitors. Int. J. Clin. Exp. Pathol., 4,403-409, 2011.

7. Nasirujjaman K., Kimura-Yoshida, C., Gotoh,N., Hiramatsu, R. and Matsuo I.: A geneticstudy of the effects of reduced Frs2alphagene dosage in exostoses development andcartilage abnormalities observed in Ext2 het-erozygous mutant mice. Journal of Osaka Medi-cal Center & Research Institute for Maternal &Child Health., 26, 101-108, 2011.

8. Gotoh, N.: FRS2. In: Encyclopedia of SignalingMolecules. Springer, Heidelberg, Germany, 2011.

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