1. The biological functions of cell-cell interac-tion in human oncogenesis

Takeshi Ito, Yumi Tsuboi, Yuki Kumagai, MasaruKasai, Atsuko Nakamura, Toko Funaki, YukiAzuma, Yoshiaki Kanamura, Yota Mitobe, Tomo-ko Masuda, Hiromi Ichihara, Kaoru Kiguchi,Motoi Oba1, Daisuke Matsubara and YoshinoriMurakami: 1Research Institute of Molecular andCell Biology of Cancer, Showa University,

Disruption of cell adhesion is a critical step to in-vasion and metastasis of human cancer and theiracquired resistance to several anti-cancer and mo-lecular targeting drugs. CADM1/TSLC is an immu-noglobulin superfamily cell adhesion molecule (Ig-CAMs) and acts as a tumor suppressor in variouscancers. By contrast, CADM1 rather promotes cellinvasion and metastasis in adult T-cell leukemia(ATL) or small cell lung cancer (SCLC). In order toelucidate molecular pathways involved in thisCADM1-mediated dual functions in oncogenesis,mass spectrometry (MS) analysis was performed toidentify a series of proteins associated with CADM1in epithelial cells and SCLC. In epithelial cells, wehave identified several molecules in the tyrosine

kinase pathways and found that interaction ofCADM1 with these tyrosine kinases could modifythe growth associated signaling triggered by tyro-sine kinases. In colon cancer, a molecule identifiedby MS analysis showed critical roles in cell mobilityand tumorigenicity in nude mice of colon cancercells through interrupting tyrosine-kinase path-ways. In SCLC, MS analysis identified a series ofproteins also important for human tumorigenesisand possibly for response against anti-cancer drugs.We are also investigating possible cross-talk of

IgCAMs and its biological and immunological sig-nificance comprehensively by cloning more than300 IgCAMs expressed in human cells and analyz-ing molecule-molecule interactions using the sur-face plasmon resonance imaging (SPRi) and the am-plified luminescence proximity homogenous assay(ALPHA). As a pilot study using SPRi approach,we quantified the affinity of interaction of CADM1with other known IgCAM molecules. The cell adhe-sion molecule (CADM) family of the immunoglobu-lin superfamily (IgSF) comprises four members,CADM1－CADM4, and participates in the forma-tion of epithelial and synaptic adhesion throughcell－cell homophilic and heterophilic interactions.To identify the partners interacting with each mem-

Department of Cancer Biology

Division of Molecular Pathology人癌病因遺伝子分野

Professor Yoshinori Murakami, M.D., Ph.D.Project Professor Takayuki MorisakiAssistant Professor Takeshi Ito, Ph.D.

教 授 医学博士 村 上 善 則特任教授 医学博士 森 崎 隆 幸助 教 博士（医学） 伊 東 剛

Human cancers develop and progress toward malignancy through accumulation ofmultiple genetic and epigenetic alterations. Elucidation of these alterations is es-sential to provide molecular targets for prevention, diagnosis, and treatment ofcancer. Our current interest is to understand the roles of cell-cell interaction in in-vasion, metastasis and immunological responses of cancer. Genomic and epi-genomic abnormalities involved in human tumors, including adult T-cell leukemia,cholangiocarcinoma, lung, breast, head and neck and urological cancers, and vari-ous common diseases are also being investigated.

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ber of the CADM family proteins, we set up a plat-form for multiple detection of the extracellular pro-tein－protein interactions using SPRi and analyzedthe interactions between the CADM family proteinsand 10 IgSF of their structurally related cell adhe-sion molecules. SPRi analysis identified a new in-teraction between CADM1 and CADM4, where thisheterophilic interaction was shown to be involvedin morphological spreading of adult T-cell leukemiacells expressing CADM1 when incubated on CADM4-coated glass. Moreover, class-I MHC-restricted T-cell-associated molecule (CRTAM) was identified toshow the highest affinity to CADM1 among itsbinding partners by comparing the dissociationconstants calculated from the SPR sensorgrams.These results suggest that the SPRi platform wouldprovide a novel screening tool to characterize extra-cellular protein－protein interactions among cellsurface- and secreted proteins, including IgSF mole-cules (1, 12).

2. Studies for establishing novel diagnostic andtherapeutic approaches to a subset of humancancer

Takeshi Ito, Ayaka Sato, Yuki Kumagai, YumiTsuboi, Tomoko Masuda, Daisuke Matsubara,Zenichi Tanei2, Masako Ikemura2, Toshio Niki3,Yasuyuki Seto4, Masashi Fukayama2 and Yoshi-nori Murakami: 2Department of Pathology and4Department of Breast and Endocrine Surgery,Graduate School of Medicine, The University ofTokyo, 3Department of Integrative Pathology, JichiMedical University

CADM1 is overexpressed in adult T-cell leukemia(ATL) and small cell lung cancer (SCLC), conferringhighly invasive or metastatic phenotypes character-istic to ATL or SCLC. To establish sensitive diag-nostic tools of ATL or SCLC through detectingCADM1, monoclonal antibodies against the frag-ments of CADM1 overexpressed in ATL or SCLCare being generated and characterized in collabora-tion with scientists in the Institute of Advanced Sci-ence and Technology, the University of Tokyo. De-tection systems of ATL and SCLC by these antibod-ies are being validated using the serum from pa-tients of ATL and SCLC in collaboration with clini-cal oncologists in the University of Tokyo Hospitaland National Cancer Research Center Hospital.These antibodies would be also promising to gener-ate several therapeutic approaches, including radio-isotope-conjugated antibodies and chimeric antigen

receptor-T cell therapy.Circulating tumor DNA (ctDNA) was also ana-

lyzed in plasma from patients of early-stage breastcancer surgically resected at the University of To-kyo Hospital, Tokyo, Japan using the PICK3CA mu-tation as an indicator (2). Similar analysis was car-ried out to establish a diagnostic approach to early-stage or early-phase recurrence of thyroid cancerusing V600E mutation of the BRAF gene. Compara-tive analysis of pre- and post-operative plasm fromthyroid cancer patients would provide a promisinginformation to predict locally invasive phenotypeand recurrence of thyroid cancer.To unveil additional molecular mechanisms un-

derlying multistage carcinogenesis, genomic, epi-genomic, and transcriptional alterations in keymolecules, including CADM1, in human tumori-genesis were examined in breast and other cancersin collaboration with others (3, 4). In lung carcino-genesis, emphysema appears to be a kind of pre-cancerous lesion and we identified that loss ofCADM1 function is involved in emphysema forma-tion in collaboration with others (5).

3. Genomic-epidemiological studies of varioushuman diseases on the basis of Biobank Ja-pan.

Yoshinori Murakami, Takayuki Morisaki, DaisukeMatsubara, Zenichi Tanei2, Makoto Hirata5 andKoichi Matsuda5: 5Laboratory of Genome Technol-ogy and 6Project Division of International Ad-vanced Medical Research, The Institute of MedicalScience, The University of Tokyo

A large number of genomic DNA from normalperipheral lymphocytes as well as serum samplesfrom more than 267,000 cases with 51 diseases wascollected and preserved in BioBank Japan in the In-stitute of Medical Science, the University of Tokyo.These DNA samples and clinical information wereshown to be valuable for genome-wide associationstudy (GWAS) of susceptibility of various humandiseases, including gastric cancer, colorectal cancer,ovarian cancer, hypospadias and diabetes, in col-laboration with a large study group of order-mademedicine and basic and clinical geneticists in Japan(6-10, 13, 14). Serum samples collected from a largenumber of patients and collected in Biobank Japanwas also shown to be in high quality for variousproteomic and metabolomic analyses in collabora-tion with others (11).

Publications

1. Ito T., Kasai Y., Kumagai Y., Suzuki D., Ochiai-Noguchi M., Irikura D., Miyake S., Murakami

Y. Quantitative analysis of interaction betweenCADM1 and its binding cell-surface proteins

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using surface plasmon resonance imaging.Frontiers in Cell and Developmental Biology 6:86, 2018.

2. Sato A., Tanabe M., Tsuboi Y., Ikemura M.,Tada K., Seto Y. and Murakami Y. Detection ofthe PIK3CA mutation in circulating tumor DNAas a possible predictive indicator for poor prog-nosis of early-stage breast cancer. Journal ofCancer Therapy, 9: 42-54, 2018.

3. Saito M, Go.to A., Abe N., Saito K., Maeda D.,Ohtake T., Murakami Y. and Takenoshita S. De-creased expression of CADM1 and CADM4 areassociated with advanced stage breast cancer.Oncology Letters, 15: 2401-2406, 2018.

4. Kanke Y., Saito M., Abe N., Saito K., Goto A.,Ohtake T., Murakami Y. and Kono K. Expres-sion profile of CADM1 and CADM4 in triplenegative breast cancer with primary systemictherapy. Oncology Letters, 17: 921-926, 2018.

5. Ri A., Hagiyama M., Inoue T., Yoneshige A.,Kimura R., Murakami Y. and Ito A. Progressionof pulmonary emphysema and continued in-crease in ectodomain shedding of cell adhesionmolecule 1 after cessation of cigarette smoke ex-posure in mice. Frontiers in Cell and Develop-mental Biology, 6: 52, 2018.

6. Yodsurang V., Tang Y., Takahashi Y., TanikawaC., Kamatani Y., Takahashi A., Momozawa Y.,Fuse N., Sugawara J., Shimizu A., FukushimaA., Hishida A., Furusyo N., Naito M., WakaiK., Yamaji T., Sawada N., Iwasaki M., TsuganeS., Hirata M., Murakami Y., Kubo M. andMatsuda K. Genome-wide association study(GWAS) of ovarian cancer in Japanese pre-dicted regulatory variants in 22q13.1. PLos One13: e0209096, 2018.

7. Suzuki K., Akiyama M., Hirata M., HorikoshiM., Hosoe J., Hozawa A., Ikeda M., Ikegawa S.,Ishigaki K., Ishigaki Y., Iwasaki M., Iwata N.,Kadota A., Kadowak. T., Kamatani Y., KanaiM., Kubo M., Kuriki K., Maeda S., Matsuda K.,Murakami Y., Naito M., Okada Y., Sasaki M.,Sawada N., Shojima N., Tannno K., Tsugane S.,Wakai K., Yamaji T., Yamamoto M. andYamauchi T. Identification of 28 novel suscepti-bility loci for type 2 diabetes in the Japanesepopulation. Nat. Genet, 27: 1486-1496, 2018.

8. Tanikawa C., Kamatani Y., Toyoshima O.,Sakamoto H., Ito H., Takahashi A., MomozawaY,, Hirata M,, Fuse N,, Takai T,, Shimizu A,,Sasaki M,, Yamaji T,, Sawada N,, Iwasaki M,,Tsugane S,, Naito M,, Hishida A,, Wakai K,,Furusyo N,, Murakami Y,, Nakamura Y,, ImotoI,, Inazawa J,, Oze I,, Sato N,, Tanioka F,,Sugimura H,, Hirose H,, Yoshida T,, Matsuo K,,Kubo M, and Matsuda K. A GWAS identifies

gastric cancer susceptibility loci at 12q24.11-12and 20q11.21. Cancer Science 109: 4015-4024,2018.

9. Kojima Y., Koguchi T., Mizuno K., Sato Y.,Hoshi S., Hata J., Nishio H., Hashimoto D.,Matsushita S., Suzuki K., Miyagawa S., Hui C.C., Tanikawa C., Murakami Y., Yamada G.,Hayashi Y. and Matsuda K. Single nucleotidepolymorphisms of HAAO and IRX 6 genes asrisk factors for hypospadias. J Urol, 201: 386-392, 2018.

10. Tanikawa C., Kamatani Y., Takahashi A.,Momozawa Y., Leveque K., Nagayama S.,Mimori K., Mori M., Ishii H., Inazawa J., FuseN., Takai-Igarashi T., Shimizu A., Sasaki M.,Yamaji T., Sawada N., Iwasaki M., Tsugane S.,Naito M., Hishida A., Wakai K., Furusyo N.,Yuji K., Murakami Y., Kubo M. and Matsuda K.GWAS identifies two novel colorectal cancerloci at 16q24.1 and 20q13.12. Carcinogenesis, 39,652-660, 2018.

11. Kabe Y., Suematsu M., Sakamoto S., Hirai M.,Koike I., Hishiki T., Matsuda A., Hasegawa Y.,Tsujita K., Ono M., Minegishi N., Hozawa A.,Murakami Y., Kubo M., Itonaga M. and HandaH. ExoCounter, a high-sensitive device forcounting the number of disease - specificexosomes in human sera. Clinical Chemistry,64: 1463-1473, 2018.

12. Ito T, Kumagai Y, Itano K, Maruyama T, Ta-mura K, Kawasaki S, Suzuki T, Murakami Y.Mathematical analysis of gefitinib resistance oflung adenocarcinoma caused by MET amplifica-tion. Biochem Biophys Res Commun, in press.

13. Tanikawa C, Kamatani Y, Terao C, Usami M,Takahashi A, Momozawa Y, Suzuki S, Ogi-shima S, Shimizu A, Satoh M, Matsuo K, Mi-kami H, Naito M, Wakai K, Yamaji T, SawadaN, Iwasaki M, Tsugane S, Kohri K, Yu ASL,Yasui T, Murakami Y, Kubo M, Matsuda K.Identification of novel urolithiasis GWAS loci ina Japanese population. J American SocietyNephrology, in press.

14. Nakatochi M, Kanai K, Nakayama A, HishidaA, Kawamura Y, Ichihara S, Akiyama M, Ike-zaki H, Furusyo N, Shimizu S, Yamamoto K,Hirata M, Okada R, Kawai S, Kawaguchi M,Nishida Y, Shimanoe C, Matsuo K, NakamuraY, Mikami H, Tamura T, Nakashima H, Naka-mura T, Kato N, Matsuda K, Murakami Y,Matsubara T, Naito M, Kubo M, Kamatani Y,Shinomiya N, Yokota M, Wakai K, Okada Yand Matsuo H. Genome-wide meta-analysisidentifies multiple novel loci associated with se-rum uric acid levels in Japanese individuals.Communications Biology, in press.

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1. Molecular mechanism of the regulation of NF-kB transcription factor

Jin Gohda1, Takao Seki2, Taishin Akiyama2 andJun-ichiro Inoue: 1Center for Asian Infectious Dis-eases, IMSUT; 2RIKEN Center for Integrative Me-dical Sciences

Transcription factor NF-kB binds specifically to adecameric motif of nucleotide, kB site, and activatestranscription. The activation of NF-kB has beendemonstrated to be carried out post-translationally

upon extracellular stimuli through membrane re-ceptors such as members of the TLR/IL-1R familyand of TNFR superfamily. In canonical NF-kB path-way, NF-kB forms a complex with regulatory pro-tein, IkB, and is sequestered in the cytoplasm priorto stimulation. Upon stimulation, IkB is rapidlyphosphorylated on two specific serine residues byIkB kinase (IKK) complex followed by lysine 48(K48)-linked ubiquitination and proteasome-de-pendent degradation of IkB. NF-kB subsequentlytranslocates to the nucleus to activate transcriptionof target genes. This project is to identify molecules

Department of Cancer Biology

Division of Cellular and Molecular Biology分子発癌分野

Professor Jun-ichiro Inoue, Ph.D.Associate Professor Takeharu Sakamoto, Ph.D.Assistant Professor Yuu Taguchi, Ph.D.Assistant Professor Mizuki Yamamoto, Ph.D.

教 授 薬学博士 井 上 純一郎准教授 博士（医学） 坂 本 毅 治助 教 博士（医学） 田 口 祐助 教 博士（医学） 山 本 瑞 生

Gene expression is largely regulated by signal transduction triggered by variousstimulations. Several lines of evidence indicate that genetic defects of moleculesinvolved in the signal transduction or the gene expression lead to abnormal celldifferentiation or tumor formation. Our goal is to understand the molecular mecha-nisms of disease pathogenesis and oncogenesis by elucidating normal regulationof intracellular signal transduction and gene expression involved in cell prolifera-tion and differentiation. We have identified and been interested in Tumor necrosisfactor receptor-associated factor 6 (TRAF6), which acts as an E3 ubiquitin ligaseto generate Lys63-linked polyubiquitin chains that are crucial for transducing sig-nals emanating from the TNFR superfamily or the TLR/IL-1R family leading to acti-vation of transcription factor NF-kB and AP-1. By generating TRAF6-deficientmice, we found that TRAF6 is essential for osteoclastogenesis, immune self-toler-ance, lymph node organogenesis and formation of skin appendices. We are cur-rently focusing on molecular mechanisms underlying TRAF6-mediated activationof signal transduction pathways and how TRAF6 is involved in osteoclastogenesisand self-tolerance. In addition, NF-kB is constitutively activated in various cancercells and this activation is likely involved in the malignancy of tumors. Thus, weare also investigating the molecular mechanisms of the constitutive activation ofNF-kB and how this activation leads to the malignancy of breast cancers andadult T cell leukemia (ATL). In addition, we are investigating novel molecularmechanisms how tumor microenvironments and inflammation are regulated.

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that regulate signal from membrane receptors toNF-kB/IkB complex. We have previously identifiedupstream activators of NF-kB, tumor necrosis factorreceptor-associated factor (TRAF) 6. TRAF6 containsRING domain in the N-terminus and acts as an E3ubiquitin-ligase to catalyze the lysine 63 (K63)-linked polyubiquitination of several signaling mole-cules and TRAF6 itself. To understand the molecu-lar mechanisms of TRAF6-mediated NF-kB activa-tion, we try to identify proteins that are ubiquiti-nated by TRAF6 upon stimulation. We took advan-tage of using the peptide that specifically bindsK63-linked polyubiquitin chain to purify such pro-teins. We have confirmed that the peptide-based af-finity column is useful for specific concentration ofrecombinant K63-linked polyubiquitin chain, sug-gesting that it also works for purification of theproteins of our interest. We are also interested innoncanonical NF-kB pathway, which is crucial forimmunity by establishing lymphoid organogenesisand B-cell and dendritic cell (DC) maturation. RelBis a major NF-kB subunit in the pathway. To eluci-date the mechanism of the RelB-mediated immunecell maturation, a precise understanding of the rela-tionship between cell maturation and RelB expres-sion and activation at the single-cell level is re-quired. Therefore, we generated knock-in mice ex-pressing a fusion protein between RelB and fluores-cent protein (RelB-Venus) from the Relb locus. TheRelbVenus/Venus mice developed without any abnormali-ties observed in the Relb－/－ mice, allowing us tomonitor RelB-Venus expression and nuclear locali-zation as RelB expression and activation. RelbVenus/VenusDC analyses revealed that DCs consist of RelB－,RelBlow and RelBhigh populations. The RelBhigh popula-tion, which included mature DCs with projections,displayed RelB nuclear localization, whereas RelBin the RelBlow population was in the cytoplasm. Al-though both the RelBlow and RelB－ populationsbarely showed projections, MHC II and co-stimula-tory molecule expression were higher in the RelBlow

than in the RelB－ splenic conventional DCs. Takentogether, our results identify the RelBlow populationas a possible novel intermediate maturation stageof cDCs and the RelbVenus/Venus mice as a useful tool toanalyze the dynamic regulation of the non-canoni-cal NF-kB pathway.

2. Molecular mechanism of RANK signaling inosteoclastogenesis

Yuu Taguchi, Yui Iwamae, Yuki Nakano, MikakoSuzuki, Saya Bando, Youko Hirayama, Jin Goh-da1, and Jun-ichiro Inoue

Bone is an important organ, which supports bodystructure and hematopoiesis. Osteoclasts are largemultinucleated cells, which have ability to degradebone matrixes, and play a crucial role in bone ho-

meostasis in concert with osteoblast, which gener-ates bone matrix. As a result of excess formation oractivation of osteoclasts, pathological bone resorp-tion is observed in postmenopausal osteoporosis,rheumatoid arthritis and bone metastasis. There-fore, elucidating the molecular mechanism of osteo-clastogenesis is important for understanding bonediseases and developing novel strategies to treatsuch diseases. Osteoclasts are differentiated fromhematopoietic stem cells upon stimulation withmacrophage colony-stimulating factor (M-CSF) andreceptor activator of NF-kB ligand (RANKL). It isknown that the activation of signal transductionpathway emanating from receptor RANK is essen-tial for osteoclastogenesis. The RANK signal activa-tes transcriptional factors, NF-kB and AP-1, throughthe E3 ubiquitin ligase TRAF6, and induces activa-tion of PLCg2-mediated Ca2＋ signaling pathway.These signals lead to the induction of NFATc1, amaster transcriptional factor in osteoclastogenesis.We have previously demonstrated that RANK hasa functional amino acid sequences, named HighlyConserved domain in RANK (HCR), which doesnot have any homology of amino-acid sequencewith other proteins. The HCR acts as a platform forformation of signal complex including TRAF6,PLCg2 and adaptor protein Gab2. This formation ofsignal complex is involved in sustaining activationof RANK signaling, and is essential for the NFATc1induction and osteoclastogenesis. To elucidate otherfunctions and the precise molecular mechanism ofHCR, we have performed yeast two-hybrid screen-ing and protein-array to identify the interactingprotein to receptor RANK including HCR. Somecandidate proteins were associated with RANK andHCR, and were involved in the induction of osteo-clast-specific gene expression, suggesting that HCRhas an additional function other than NFATc1 in-duction. We are currently investigating the molecu-lar mechanisms of these candidate proteins in os-teoclastogenesis. Moreover, to reveal the novelmechanisms involved in osteoclastogenesis, we per-formed microarray analysis of gene expression lev-els during osteoclastogenesis. Since some geneswere dramatically downregulated in response toRANKL stimulation, we are currently investigatingwhether these genes are involved in the regulationof osteoclastogenesis in vitro and in vivo. We alsoobtained candidate genes involved in osteoclasto-genesis by using CRISPR/Cas9-gRNA library scree-ning system. Furthermore, to develop drugs fortreatment to osteoporosis, we tried to search candi-date compounds which have an ability to suppressosteoclastogenesis. Because some compoundsshowed inhibitory effect to osteoclastogenesis in vi-tro without arresting cell growth, we are now try-ing to elucidate mechanisms of inhibition.

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3. Intratumoral bidirectional transitions betweenepithelial and mesenchymal cells in triple-negative breast cancer

Mizuki Yamamoto, Chiho Abe, Aya Watanabeand Jun-ichiro Inoue

Epithelial-mesenchymal transition (EMT) and itsreverse process, MET, are crucial in several stagesof cancer metastasis. EMT allows cancer cells tomove to proximal blood vessels for intravasation.However, because EMT and MET processes are dy-namic, mesenchymal cancer cells are likely to un-dergo MET transiently and subsequently re-un-dergo EMT to restart the metastatic process. There-fore, spatiotemporally-coordinated mutual regula-tion between EMT and MET could occur duringmetastasis.To elucidate such regulation, we chose HCC38, a

human triple-negative breast cancer cell line, be-cause HCC38 is composed of epithelial and mesen-chymal populations at a fixed ratio even thoughmesenchymal cells proliferate significantly moreslowly than epithelial cells. We established E-cad-herin- and Vimentin-reporter expressing HCC38cells to analyze EMT status using FACS analysisand live cell imaging. Using this cell, we performedCRISPR/Cas9-mediated screening for intratumoralEMT-regulating genes and found several candi-dates.

4. Molecular mechanism of the Flavi virus E-pro-tein-mediated membrane fusion.

Mizuki Yamamoto, Yusuke Fujinami, Aya Wata-nabe and Jun-ichiro Inoue

We have developed a cell-based fusion assay forprME protein of Flavi virus in a low pH-dependentmanner, using Aedes albopictus cell line C6/36 cellsexpressing Renilla luciferase (RL)-based split re-porter proteins. Using this assay, we are investigat-ing molecular mechanisms for the E-protein-medi-ated membrane fusion.

5. Mint3 depletion attenuates proliferation inpancreatic cancer cells

Akane Kanamori, Jun-ichiro Inoue and TakeharuSakamoto

Pancreatic cancer is one of the deadliest cancers.Although severe hypoxia is characteristic for pan-creatic cancer, most cancer cells grow in normoxicand modest hypoxic areas. Hypoxia inducible fac-tor-1 (HIF-1) is a master transcriptional factor forhypoxic response and thought to promote the ma-lignancy of pancreatic cancer not only in severe hy-poxic area but also in normoxic and modest hy-

poxic areas where cancer cells grow. However, theimportance of HIF-1 in pancreatic cancer prolifera-tion under the normoxic condition remains unclear.To address this, we focused on Mint3 which acti-vates HIF-1 even in normoxia in cancer cells. Mint3depletion attenuated proliferation in human pancre-atic cancer AsPC1, BxPC3, Panc-1, and MIA-PaCa2cells. Further analyses revealed that Mint3 deple-tion caused cell cycle arrest with increased p21 andp27 expression in pancreatic cancer cells in a HIF-1-dependent manner. Mint3 depletion also attenuatedorthotopic tumor growth of AsPC1 cells in immu-nodeficient mice. Thus, Mint3 is a possible targetfor pancreas cancer treatment.

6. Pathophysiological analyses of the genes re-lated to oxygen-dependent cancer cell prolif-eration using genetically modified mice

Yuya Fukui, Yoohwa Chung, Jun-ichiro Inoue andTakeharu Sakamoto

Responses to outside stimuli are essential forbody homeostasis and dysregulation of these re-sponses can cause pathological conditions such asinflammatory diseases and cancer. We previouslysurveyed the genes related to oxygen-dependentcancer cell proliferation using genome-wide shRNAlibraries and identified 13 genes. Among them, wefurther focused on two genes, X and Y, whosepathophysiological roles remain unclear and estab-lished conventional and conditional knockout miceof these genes. Gene X was expressed ubiquitouslyin adult mouse tissues and Gene X knockout miceshowed embryonic lethality. Interestingly, depletionof Gene X in myeloid cells promoted acute inflam-mation in TPA-induced ear inflammation, DSS-in-duced colitis, and LPS-induced septic shock modelswithout affecting myeloid cell differentiation in thesteady state. Meanwhile, Gene Y was predomina-ntly expressed in testes among adult mouse tissues.Gene Y knockout mice were born less frequentlythan expected Mendelian ratio. Gene Y knockoutmice exhibited male infertility. Thus, Gene Y con-tributes to embryogenesis to some extent and malefertility in vivo. We further examined the role ofGene Y in the tumor microenvironment. Tamo-xifen-induced Gene Y conditional knockout miceshowed attenuated tumor growth of injectedmurine breast cancer E0771 cells compared withcontrol mice. Further analyses revealed that Gene Ywas expressed in some of CD45＋ cells in E0771 tu-mor tissues. Thus, Gene Y expression in someCD45＋ cells might affect tumor growth.

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Publications

Kajikawa, S., Taguchi, Y., Hayata, T., Ezura, Y.,Ueta, R., Arimura, S., Inoue, J., Noda, M. andYamanashi, Y. Dok-3 and Dok-1/-2 adaptors playdistinctive roles in cell fusion and proliferationduring osteoclastogenesis and cooperatively pro-tect mice from osteopenia. Biochem. Biophys. Res.Commun. 498, 967-974. (2018) doi: 10.1016/j.bbrc.2018.03.090.

Gohda, J., Suzuki, K., Liu, K., Xie, X., Takeuchi, H.,Inoue, J., Kawaguchi, Y. and Ishida, T. BI-2536and BI-6727, dual Polo-like kinase/bromodomaininhibitors, effectively reactivate latent HIV-1. Sci.Rep. 8: 3521 (2018). doi: 10.1038/s41598-018-21942-5.

Liu, D., Wang, H., Yamamoto, M., Song, J., Zhang,R., Du, Q., Kawaguchi, Y., Inoue, J. and Matsuda,Z. Six-helix bundle completion in the distal C-ter-minal heptad repeat region of gp41 is requiredfor efficient human immunodeficiency virus type1 infection. Retrovirology (2018) 15: 27. doi:10.1186/s12977-018-0410-9

Ogawa, M., Matsuda, R., Takada, N., Tomokiyo,M., Yamamoto, S., Shizukusihi, S., Yamaji, T.,Yoshikawa, Y., Yoshida, M., Tanida, I., Koike, M.,Murai, M., Morita, H., Takeyama, H., Ryo, A.,Guan, J.-L., Yamamoto, M., Inoue, J., Yanagawa,T., Fukuda, M., Kawabe, H. and Ohnishi, M.Mechanisms of Streptococcus pneumoniae-1 tar-geted autophagy via pneumolysin, Golgi-residentRab41, and Nedd4-1 mediated K63-linkedubiquitination. Cell Microbiol. (2018) 26:e12846.doi: 10.1111/cmi.12846.

Johmura, Y., Maeda, I., Suzuki, N., Wu, W., Goda,A., Morita, M., Yamaguchi, K., Yamamoto, M.,Nagasawa, S., Kojima, Y., Tsugawa, K., Inoue, N.,Miyoshi, Y., Osako, T., Akiyama, F., Maruyama,

R., Inoue, J., Furukawa, Y., Ohta, T and Nakani-shi, M. Fbxo22-mediated KDM4B degradation de-termines selective estrogen receptor modulatoractivity in breast cancer. J. Clin. Invest. (2018).https://doi.org/10.1172/JCI121679.

Hatanaka, N., Seki, T., Inoue, J., Tero, A. and Suzu-ki, T. Critical roles of IkBa and RelA phospho-rylation in transitional oscillation in NF-kB sig-naling module. J. Theor. Biol. In press (2018).

Terada, H., Saitoh, Y., Kametani, K., Sakaguchi, M.,Sakamoto, T., Kamijo, A., Terada, N.＊ Electronmicroscopic observation of photoreceptor cells indirectly inserted anesthetized Drosophila into ahigh-pressure freezing unit. Microsc Res Tech. (inpress). doi: 10.1002/jemt.23166.

Saitoh, Y., Kamijo, A., Yamauchi, J., Sakamoto, T.,Terada, N.＊ The membrane palmitoylated pro-tein, MPP6, is involved in myelin formation inthe mouse peripheral nervous system. HistochemCell Biol. (in press). doi: 10.1007/s00418-018-1745-y.

Tsubaki, T., Kadonosono, T., Sakurai, S., Shiozawa,T., Goto, T., Sakai, S., Kuchimaru, T., Sakamoto,T., Watanabe, H., Kondoh, G., Kizaka-Kondoh,S.＊ Novel adherent CD11b＋ Gr-1＋ tumor-infil-trating cells initiate an immunosuppressive tumormicroenvironment. Oncotarget 9, 11209－11226(2018). doi: 10.18632/oncotarget.24359.

Yamamoto, M., Du, Q., Song, J., Wang, H.,Watanabe, A., Tanaka, Y., Kawaguchi, Y., Inoue,J. and Matsuda, Z. Cell-cell and virus-cell fusionassay-based analyses of alanine insertion mutantsin the distal a9 portion of the JRFL gp41 subunitfrom HIV-1. J. Biol. Chem. jbc. RA118.004579.(2019) doi: 10.1074/jbc. RA118.004579.

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1. Activation of the receptor tyrosine kinaseMuSK by the cytoplasmic protein Dok-7 inneuromuscular synaptogenesis.

Eguchi, T., Ueta, R., Tezuka, T., Weatherbee, SD.1,and Yamanashi, Y.: 1Department of Genetics, YaleUniversity.

Protein-tyrosine kinases (PTKs) play crucial rolesin a variety of signaling pathways that regulateproliferation, differentiation, motility, and other ac-tivities of cells. Therefore, dysregulated PTK signalsgive rise to a wide range of diseases such as neo-plastic disorders. To understand the molecularbases of PTK-mediated signaling pathways, weidentified Dok-1 as a common substrate of manyPTKs in 1997. Since then, the Dok-family has beenexpanded to seven members, Dok-1 to Dok-7,which share structural similarities characterized byN-terminal pleckstrin homology (PH) and phospho-tyrosine binding (PTB) domains, followed by Srchomology 2 (SH2) target motifs in the C-terminalmoiety, suggesting an adaptor function. Indeed, asdescribed below, Dok-1 and Dok-2 recruit p120 ras-

GAP upon tyrosine phosphorylation to suppressRas-Erk signaling. However, we found that Dok-7acts as an essential cytoplasmic activator of themuscle-specific receptor tyrosine kinase (RTK)MuSK in the formation of the neuromuscular junc-tion (NMJ), providing a new insight into RTK-me-diated signaling. It seems possible that local levelsof cytoplasmic activators, like Dok-7, control the ac-tivity of RTKs in concert with their extracellularligands.The NMJ is a synapse between a motor neuron

and skeletal muscle, where the motor nerve termi-nal is apposed to the endplate (the region of synap-tic specialization on the muscle). The contraction ofskeletal muscle is controlled by the neurotransmit-ter acetylcholine (ACh), which is released from thepresynaptic motor nerve terminal. To achieve effi-cient neuromuscular transmission, acetylcholine re-ceptors (AChRs) must be densely clustered on thepostsynaptic muscle membrane of the NMJ. Failureof AChR clustering is associated with disorders ofneuromuscular transmission such as congenital my-asthenic syndromes and myasthenia gravis, whichare characterized by fatigable muscle weakness. The

Department of Cancer Biology

Division of Genetics腫瘍抑制分野

Professor Yuji Yamanashi, Ph.D.Assistant Professor Tohru Tezuka, Ph.D.Assistant Professor Ryo Ueta, Ph.D.Assistant Professor Sumimasa Arimura, Ph.D.Assistant Professor Akane Inoue-Yamauchi, Ph.D.

教 授 理学博士 山 梨 裕 司助 教 博士（理学） 手 塚 徹助 教 博士（生命科学） 植 田 亮助 教 博士（医学） 有 村 純 暢助 教 博士（医学） 山内（井上） 茜

The major interest of this division is in molecular signals that regulate a variety ofcellular activities. Our aim is to address how dysregulated cellular signals give riseto neoplastic, immune, neural, metabolic, or developmental disorders. Our goal isto understand the molecular bases of tumorigenesis and the development of otherintractable diseases as a path toward uncovering therapeutic targets. Currently,we are investigating regulatory mechanisms in protein-tyrosine kinase (PTK)-medi-ated signaling pathways, their pathophysiological roles and the potential for thera-peutic intervention.

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formation of NMJs is orchestrated by MuSK and byneural agrin, an extracellular activator of MuSK.However, experimentally when motor nerves areablated, AChRs form clusters in the correct, centralregion of muscle during embryogenesis in a MuSK-dependent process known as prepatterning of thereceptors. In addition, in vivo overexpression ofMuSK causes neuromuscular synapse formation inthe absence of agrin, suggesting that muscle-intrin-sic, cell-autonomous activation of MuSK may beadequate to trigger presynaptic and postsynapticdifferentiation in vivo. However, the mechanismsby which MuSK is activated independently of nerveand agrin had long been unclear.Because both MuSK and the adaptor-like cyto-

plasmic protein Dok-7 are localized to the postsyn-aptic region of NMJs, we examined their interactionand found that Dok-7 is an essential cytoplasmicactivator of MuSK. In addition, we found thatDok-7 directly interacts with the cytoplasmic por-tion of MuSK and activates the RTK, and that neu-ral agrin requires Dok-7 in order to activate MuSK.Indeed, in vivo overexpression of Dok-7 increasedMuSK activation and promoted NMJ formation.Conversely, mice lacking Dok-7 formed neitherNMJs nor AChR clusters. In addition, we have re-cently found that postnatal knockdown of dok-7gene expression in mice causes structural defects inNMJs and myasthenic pathology, suggesting an es-sential role for Dok-7 not only in the embryonicformation but also in the postnatal maintenance ofNMJs.Interestingly, mice lacking Lrp4, which forms a

complex with MuSK and acts as an essential agrin-binding module, do not show MuSK-dependentAChR prepatterning or NMJ formation. This sug-gests that Lrp4 is required for MuSK activation un-der physiological conditions, in contrast to our ob-servation that Dok-7 can activate MuSK in the ab-sence of Lrp4 or its ligand agrin, at least in vitro.Thus, we examined the effects of forced expressionof Dok-7 in skeletal muscle on NMJ formation inthe absence of Lrp4 and found that it indeed in-duces MuSK activation in mice lacking Lrp4. How-ever, the activation level of MuSK was significantlylower in the absence than in the presence of Lrp4.Together, these data indicate that Lrp4 is requiredfor efficient activation of MuSK by Dok-7 in themuscle. Since Lrp4 is also essential for presynapticdifferentiation of motor nerve terminals in the em-bryonic NMJ formation (Nature 489: 438-442, 2012),this apparent cooperation between Lrp4 and Dok-7in MuSK activation may be complicated.Although we previously failed to detect MuSK

activation in cultured myotubes by Dok-7 that lacksthe C-terminal region (Dok-7-DC), we have recentlyfound that purified, recombinant Dok-7-DC showsmarginal ability to activate MuSK's cytoplasmicportion, carrying the kinase domain. Consistently,

forced expression of Dok-7-DC rescued Dok-7knockout mice from neonatal lethality caused bythe lack of NMJs, indicating restored MuSK activa-tion and NMJ formation. However, these miceshowed only marginal activation of MuSK and diedby 3 weeks of age apparently due to an abnormallysmall number and size of NMJs. Therefore, Dok-7'sC-terminal region plays a key, but not fully essen-tial, role in MuSK activation and NMJ formation.Interestingly, overexpression of Dok-7 in skeletal

muscle abnormally activates MuSK, leading to theformation of abnormally large NMJs in mice. How-ever, these mice with abnormally large NMJs showno obvious motor dysfunction. We are investigatingelectrophysiological and ultrastructural propertiesof the abnormally large NMJs in vivo.

2. Agrin's role aside from MuSK activation in thepostnatal maintenance of NMJs.

Tezuka, T., Burgess, RW.1, Ueta, R., and Yama-nashi, Y.: 1The Jackson Laboratory.

Although NMJ formation requires agrin underphysiological conditions, it is dispensable for NMJformation experimentally in the absence of the neu-rotransmitter acetylcholine, which inhibits postsyn-aptic specialization. Thus, it was hypothesized thatMuSK needs agrin together with Lrp4 and Dok-7 toachieve sufficient activation to surmount inhibitionby acetylcholine. To test this hypothesis, we exam-ined the effects of forced expression of Dok-7 inskeletal muscle on NMJ formation in the absence ofagrin and found that it indeed restores NMJ forma-tion in agrin-deficient embryos. However, theseNMJs rapidly disappeared after birth, whereas ex-ogenous Dok-7-mediated MuSK activation wasmaintained. These findings indicate that the MuSKactivator agrin plays another role essential for thepostnatal maintenance, but not for embryonic for-mation, of NMJs. Because a pathogenic mutation ofagrin in patients with congenital myasthenic syn-dromes (see below) did not show impaired abilityto activate MuSK at least in vitro (Am. J. Hum.Genet., 85: 155-167, 2009), the novel role of agrinmay be relevant to pathogenicity of the mutation.We are investigating minimal regions of agrin re-quired for the role to understand molecular mecha-nisms underlying the agrin-mediated postnatalmaintenance of NMJs.

3. Pathophysiological mechanisms underlyingDOK7 myasthenia.

Eguchi, T., Saito A., Ueta R., Tezuka, T., Arimu-ra, S., Fukudome T.1, Sagara H.2, Motomura, M.3,Yoshida, N.4, Beeson, D.5, and Yamanashi, Y.: 1De-partment of Neurology, Nagasaki Kawatana Medi-cal Center. 2Medical Proteomics Laboratory, IM-

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SUT. 3Department of Engineering, Faculty of En-gineering, Nagasaki Institute of Applied Science.4Laboratory of Developmental Genetics, IMSUT.5Weatherall Institute of Molecular Medicine, Uni-versity of Oxford.

As mentioned above, impaired clustering ofAChRs could underlie NMJ disorders, be they auto-immune (MuSK antibody-positive myasthenia gra-vis) or genetic (congenital myasthenic syndromes(CMS)) in origin. Therefore, our findings that Dok-7activates MuSK to cluster AChRs and to form NMJssuggested DOK7 as a candidate gene for mutationsassociated with CMS. Indeed, we demonstrated thatbiallelic mutations in DOK7 underlie a major sub-group of CMS with predominantly proximal mus-cle weakness that did not show tubular aggregateson muscle biopsy but were found to have normalAChR function despite abnormally small and sim-plified NMJs. We further demonstrated that severalmutations, including one associated with the major-ity of patients with the disease, impaired Dok-7'sability to activate MuSK. This new disease entity istermed "DOK7 myasthenia."To investigate pathophysiological mechanisms

underlying DOK7 myasthenia, we establishedknock-in mice (Dok-7 KI mice) that have a muta-tion associated with the majority of patients withDOK7 myasthenia. As expected, Dok-7 KI miceshowed characteristic features of severe muscleweakness and died between postnatal day 13 and20. Furthermore, they showed abnormally smallNMJs lacking postsynaptic folding, a pathologicalfeature seen in patients with DOK7 myasthenia.Consistent with this, Dok-7 KI mice exhibited de-creased MuSK activity in skeletal muscle, indicatingthat the Dok-7 KI mice develop defects similar tothose found in patients with DOK7 myasthenia, al-though the mice exhibit a more severe phenotype.We are investigating other defects in NMJ functionsand detailed pathophysiology, including electro-physiology and ultrastructural physiology in theDok-7 KI mice with or without potential therapeu-tic treatments.

4. DOK7 gene therapy that enlarges NMJs.

Arimura, S., Ueta, R., Kanno, T., Tezuka, T.,Okada, H.1, Kasahara, Y.1, Tomono, T.1, Moto-mura, M.2, Yoshida, N.3, Beeson, D.4, Takeda, S.5,Okada, T.1, and Yamanashi, Y.: 1Department ofBiochemistry and Molecular Biology, NipponMedical School. 2Department of Engineering, Fac-ulty of Engineering, Nagasaki Institute of AppliedScience. 3Laboratory of Developmental Genetics,IMSUT. 4Weatherall Institute of Molecular Medi-cine, University of Oxford. 5Department of Mo-lecular Therapy, National Institute of Neurosci-ence.

As mentioned above, DOK7 myasthenia is associ-ated with impaired NMJ formation due to dec-reased ability of Dok-7 to activate MuSK in myo-tubes at least in part. Interestingly, in vivo overex-pression of Dok-7 increased MuSK activation andpromoted NMJ formation in the correct, central re-gion of the skeletal muscle. Because these geneti-cally manipulated mice did not show obvious de-fects in motor activity, overexpression of Dok-7 inthe skeletal muscle of patients with DOK7 myasthe-nia might ameliorate NMJ formation and muscleweakness. To test this possibility, we generated anAdeno-associated virus-based vector (AAV-D7),which strongly expressed human Dok-7 in myo-tubes and induced AChR cluster formation. Indeed,therapeutic administration of AAV-D7 to Dok-7 KImice described above resulted in enlargement ofNMJs and substantial increases in muscle strengthand life span. Furthermore, when applied to modelmice of another neuromuscular disorder, autosomaldominant Emery-Dreifuss muscular dystrophy,therapeutic administration of AAV-D7 likewise re-sulted in enlargement of NMJs as well as positiveeffects on motor activity and life span. These resultssuggest that therapies aimed at enlarging the NMJmay be useful for a range of neuromuscular disor-ders. Indeed, we have recently found that therapeu-tic administration of AAV-D7 is beneficial to othermouse models of neuromuscular disorders, includ-ing amyotrophic lateral sclerosis (ALS), a progres-sive, multifactorial motor neurodegenerative dis-ease with severe muscle atrophy. We are further in-vestigating the effects of AAV-D7 administration inother types of muscle weakness.

5. Lrp4 antibodies in patients with myastheniagravis.

Tezuka, T., Motomura, M.1, and Yamanashi, Y.:1Department of Engineering, Faculty of Engineer-ing, Nagasaki Institute of Applied Science.

Myasthenia gravis (MG) is an autoimmune dis-ease of the NMJ. About 80％ of patients with gener-alized MG have AChR antibodies, the presence ofwhich is a causative factor for the disease, and avariable proportion of the remaining patients (0-50％ throughout the world) have MuSK antibodies.However, diagnosis and clinical management re-main complicated for patients who are negative forMuSK and AChR antibodies. Given the essentialroles and postsynaptic localization of Lrp4 in theNMJ, we hypothesized that Lrp4 autoantibodiesmight be a pathogenic factor in MG. To test thishypothesis, we developed a luminescence-basedmethod to efficiently detect serum autoantibodies toLrp4 in patients, and found that 9 patients werepositive for antibodies to the extracellular portionof Lrp4 from a cohort of 300 patients with AChR

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antibody-negative MG. 6 of these 9 patients withLrp4 antibody-positive MG were also negative forMuSK antibodies, and generalized MG was diag-nosed in all 9 patients, who showed severe limbmuscle weakness or progressive bulbar palsy orboth. Thymoma was not observed in any of thesepatients, unlike the situation in patients with AChRantibody-positive MG. Furthermore, we confirmedthat serum antibodies to Lrp4 recognize its nativeform and inhibit binding of Agrin to Lrp4, which iscrucial for NMJs. Also, we found that Lrp4 autoan-tibodies were predominantly comprised of IgG1, acomplement activator, implicating the potential forthese antibodies to cause complement-mediated im-pairment of NMJs. Together, our findings indicatethe involvement of Lrp4 antibodies in the patho-genesis of AChR antibody-negative MG. Followingthis study, two groups in Germany and USA re-ported respectively that about 50％ and 10％ of MGpatients, who were negative for both MuSK andAChR antibodies, were positive for antibodies toLrp4, and that these Lrp4 antibodies inhibited agrinand MuSK-mediated AChR clustering in culturedmyotubes (J. Neurol., 259: 427-435, 2012; Arch. Neu-rol., 69: 445-451, 2012). Also it was reported that an-tibodies to Lrp4 inhibited agrin/MuSK signalingand induced MG in model animals (J. Clin. Invest.,123: 5190-5202, 2013). Thus, LRP4 antibodies appearto be a new biomarker of MG (Ann. N.Y. Acad. Sci.1413: 126-135, 2018). We are investigating patho-genicity of Lrp4 antibodies.

6. Roles of Dok-1 to Dok-6.

Inoue-Yamauchi, A., Jozawa, H., Arimura, S., Ka-jikawa, S., Kanno, T., Takasaka, K., Shimura, E.1,Hayata, T.2, Ezura, Y.3, Taguchi, Y.4, Ueta, R.,Oda, H.5, Nakae, S.1, Inoue, J.4, Yoshida, N.6,Noda, M.3, and Yamanashi, Y.: 1Laboratory ofSystems Biology, IMSUT. 2Department of Biologi-cal Signaling and Regulation, Faculty of Medicine,University of Tsukuba. 3Department of SkeletalMolecular Pharmacology, Medical Research Insti-tute, Tokyo Medical and Dental University. 4Divi-sion of Cellular and Molecular Biology, IMSUT.5Department of Pathology, Tokyo Women's Medi-cal University. 6Laboratory of Developmental Ge-netics, IMSUT.

Dok-family proteins can be classified into threesubgroups based on their structural similarities andexpression patterns; namely, 1) Dok-1, -2, and -3,which are preferentially expressed in hematopoieticcells, 2) Dok-4, -5, and -6, which are preferentiallyexpressed in non-hematopoietic cells, and 3) Dok-7,which is preferentially expressed in muscle cells. Asmentioned above, Dok-1 and its closest paralog,Dok-2, recruit p120 rasGAP upon tyrosine phos-phorylation to suppress Ras-Erk signaling. Al-

though Dok-3 does not bind with p120 rasGAP, italso inhibits Ras-Erk signaling. Consistently, wedemonstrated that Dok-1, Dok-2 and Dok-3 are keynegative regulators of hematopoietic growth andsurvival signaling. For example, Dok-1, Dok-2, andDok-3 cooperatively inhibit macrophage prolifera-tion and Dok-1－/－Dok-2－/－Dok-3－/－ mice develophistiocytic sarcoma, an aggressive malignancy ofmacrophages. Also, we found that Dok-1 andDok-2 negatively regulate intestinal inflammationin the dextran sulfate sodium-induced colitismodel, apparently through the induction of IL-17Aand IL-22 expression. However, we have recentlyfound that Dok-3 and Dok-1/-2 play distinctiveroles in cell fusion and proliferation during osteo-clastogenesis and cooperatively protect mice fromosteopenia. We are further investigating roles ofDok-1 to Dok-6, including those in tumor malig-nancy, inflammatory disorders, bone homeostasis,and other types of intractable diseases.

7. Omic analyses.

Ueta, R., Eguchi, T., Jozawa, H., Saito A., Tezuka,T., Arimura, S., Miyoshi. S., Takada Y.1, Iemura,S.2, Natsume, T.3, Kozuka-Hata, H.4, Oyama, M.4,and Yamanashi, Y.: 1Laboratory of Glyco-bioengi-neering, The Noguchi Institute, 2Translational Re-search Center, Fukushima Medical University.3National Institute of Advanced Science and Tech-nology, Molecular Profiling Research Center forDrug Discovery. 4Medical Proteomics Laboratory,IMSUT.

To gain insights into signaling mechanisms un-derlying a variety of physiological and pathophysi-ological events, including NMJ formation, tumori-genesis, and tumor metastasis, we have performedproteomic and transcriptomic analyses. We are in-vestigating the roles of candidate proteins andgenes that appear to be involved in each of thesebiological events. In addition, we have prepared ex-perimental settings for other omic approaches suchas glycomic and metabolomic analyses.For instance, we performed mass spectrometric

analysis of Lrp4-binding proteins and found thechaperon Mesdc2 as a candidate. We confirmedtheir binding in cells, and revealed that Mesdc2bind selectively to the lower molecular mass formof Lrp4 (lower Lrp4) but not to the upper, moreglycosylated form (upper Lrp4). Although theMesdc2 binds to lower Lrp4, forced expression ofMesdc2 increased upper Lrp4, implying a role forMesdc2 in the Lrp4 glycosylation, which might fa-cilitate the receptor's cell surface expression. In-deed, we found that down regulation of Mesdc2 ex-pression in cultured myotubes suppressed cell-sur-face expression of Lrp4, or upper Lrp4 more spe-cifically. Furthermore, downregulation of Mesdc2

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also inhibited agrin-induced postsynaptic speciali-zation in myotubes, which requires binding of Lrp4to its extracellular ligand, the neural agrin. To-gether, these findings demonstrated that Mesdc2plays a key role in Lrp4-dependent postsynapticspecialization probably by promoting glycosylationand cell-surface expression of Lrp4 in myotubes.We are investigating glycomic, transcriptomic andmetabolomic data from skeletal muscle in order tounderstand molecular mechanisms underlying mus-cle weakness.

8. Screening of chemical compound and siRNAlibraries.

Ueta, R., Kosuge, M., Nagatoishi, S.1, Tsumoto, K.1,

and Yamanashi, Y.: 1Medical Proteomics Labora-tory, IMSUT.

In addition to the omic analyses described above,we performed high throughput screenings ofchemical compound and siRNA libraries, aiming tointervene in pathogenic signals or to gain insightsinto signaling mechanisms underlying a variety ofbiological events. We are investigating in vivo ef-fects of hit compounds or down-regulation of can-didate genes, and continue the ongoing screeningsto further collect appropriate hit compounds andcandidate genes that may regulate important sig-nalings. We are also investigating target proteinsfor the hit compounds to understand their modesof actions.

Publications

Kajikawa S., Taguchi Y., Hayata T., Ezura Y., UetaR., Arimura S., Inoue J.I., Noda M., and Yama-nashi Y. Dok-3 and Dok-1/-2 adaptors play dis-tinctive roles in cell fusion and proliferation dur-

ing osteoclastogenesis and cooperatively protectmice from osteopenia. Biochemical and BiophysicalResearch Communications 498: 967-974 (2018)

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1. Regulatory mechanisms of aging and carcino-genesis by senescent cells in vivo

Yoshikazu Johmura, Chieko Konishi, Sayaka Ya-mane, Yoshie Chiba, Dan Li, Wang Tehwei, Take-hiro Yamanaka, Kisho Yokote, Narumi Suzuki,Shizuka Takeyama, Honoka Hagiwara, SatotakaOhmori, Tomomi Kanai, Akane Kenjo, and Ma-koto Nakanishi:

Several lines of evidences have underpinned aprominent role of senescent cells in aging, life spanand pathogenesis of age-associated disorders. Dura-ble cessation of proliferation in response to anygrowth signals is an essential hallmark of senes-cence, but other characters including apoptosis re-sistance, mitochondrial dysfunction, and secretoryphenotypes (SASP) are also notable. In addition tothe above physiological aspects, senescent cellsshowed various unique morphological peculiarityin metabolic organelles. These morphological char-acters are typically observed independent of senes-cence-inducting stimuli and thus might underliecommon metabolic reorganizations that ensure theirsurvival and physiological traits.

Accumulation of senescent cells strongly associ-ates with a variety of age-related pathologies, suchas atherosclerosis, type II diabetes, and cancers. Re-cently, selective elimination of p16-positive senes-cent cells (senolysis) by transgenic approach fromprogeroid and normally aged mice has been re-ported to improve the healthy lifespan and amelio-rates the consequences of various age-related disor-ders. Because of the characteristic heterogeneity ofsenescence in vivo depending on a variety of the in-ducing factors such as DNA damage, oncogene ac-tivation, oxidative stress as well as telomere short-ening, the compounds and molecular targets thateffectively induce a lethality into all types of senes-cent cells have not yet been identified. In order todevelop such drugs, we uncovered the metabolicvulnerability in senescence in which glutaminolysiswas addicted for lysosome-mediated intracellularlow pH. Inhibition of glutaminolysis effectively in-duced senolysis both in vitro and in vivo.

2. Fbxo22 as a prognostic value for luminal A-type breast cancers

Yoshikazu Johmura, Narumi Suzuki, Chieko Ko-

Department of Cancer Biology

Division of Cancer Cell Biology癌防御シグナル分野

Professor Makoto Nakanishi, M.D., Ph.D.Associate Professor Atsuya Nishiyama, Ph.D.Assistant Professor Yoshikazu Johmura, Ph.D.

教 授 医学博士 中 西 真准教授 博士（理学） 西 山 敦 哉助 教 博士（薬学） 城 村 由 和

In response to genetic and epigenetic insults, normal human cells execute variouscellular responses such as transient cell cycle arrest, apoptosis, and cellular se-nescence as an anti-tumorigenesis barrier. Our research interests are to elucidatethe mechanisms underlying these cellular responses. On the basis of thesemechanisms, our final goal is to develop innovative cancer therapies and preven-tion. We are currently working on regulatory mechanisms of senescence and theirimplications in aging and carcinogenesis in vivo. Mechanisms underlying mainte-nance of genomic and epigenomic integrities such as DNA methylation mainte-nance and spindle assembly checkpoints are also under investigation.

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nishi, Sayaka Yamane, Yoshie Chiba, Dan Li,Wang Tehwei, Takehiro Yamanaka, Kisho Yoko-te, Shizuka Takeyama, Honoka Hagiwara, Sato-taka Ohmori, Tomomi Kanai, Akane Kenjo, To-mohiko Ohta1, and Makoto Nakanishi: 1Depart-ment of Translational Oncology, St. MariannaUniversity Graduate School of Medicine

Breast cancer is the most frequently diagnosedcancer in women. Approximately 70％ of breastcancers are positive for estrogen receptor-a (ER),and tamoxifen (TAM) is the standard drug fortreatment of ER-positive breast cancer, especiallyfor premenopausal women. Treatment with TAMas an adjuvant decreases the annual breast cancermortality by approximately 30％. However, up to25％ of patients with early stage breast cancertreated with TAM experience relapse of the diseasewithin 15 years. Hence, modification of the treat-ment is absolutely required for some populations.We demonstrated an unidentified series of regula-tory mechanisms controlling cofactor dynamics onER and TAM function whose activities require F-box protein 22 (Fbxo22). Skp, Cullin, F-box contain-ing complex (SCF)Fbxo22 ubiquitylates lysine demeth-ylase 4B (KDM4B) complexed with TAM-bound ER,whose degradation releases steroid receptor coacti-vator (SRC) from ER. Depletion of Fbxo22 results inER-dependent transcriptional activation via transac-tivation function 1 (AF1) function even in the pres-ence of SERMs. In living cells, tamoxifen releasesSRC and KDM4B from ER in a Fbxo22-dependentmanner. SRC release by tamoxifen requires Fbxo22on almost all ER-SRC-bound enhancer/promoters.Tamoxifen fails to prevent growth of Fbxo22-de-pleted ER-positive breast cancers both in vitro andin vivo. Clinically, a low level of Fbxo22 in tumortissues predicts a poorer outcome in ER-positive/Human Epidermal Growth Factor Receptor Type 2(HER-2)-negative breast cancers with high hazardratios independently of other markers such as Ki-67and node status. We propose that the level ofFbxo22 in tumor tissues defines a new subclass ofER-positive breast cancers for which patientsSCFFbxo22-mediated KDM4B degradation can be atherapeutic target by the next generation of SERMs.

3. Regulation of maintenance DNA methylationby dual-mono ubiquitylated PAF15

Atsuya Nishiyama, Chieko Konishi, Yoshie Chiba,Soichiro Kumamoto, Ryota Miyashita, TomomiNagatani, Kyohei Arita1, Heinrich Leonhardt2, andMakoto Nakanishi: 1Graduate School of MedicalLife Science, Yokohama City University, 2Depart-ment of Biology II and Center for Integrated Pro-tein Science Munich, Ludwig-Maximilians-Univer-sity of Munich

DNA cytosine methylation is a conserved epige-netic modification essential for embryonic develop-ment, transcriptional regulation, and genome stabil-ity. In higher eukaryotes, individual differentiatedcells possess unique DNA methylation patterns thatdetermine their own cellular phenotypes. Therefore,the DNA methylation pattern must be preciselymaintained in coordination with DNA replicationduring S phase. We and others have recently re-ported that UHRF1-mediated dual mono-ubiq-uitylation of histone H3 (H3Ub2) on lysine residues14, 18 and 23 plays a role in the recruitment ofDNMT1 and its enzymatic activation, ensuring thehigh fidelity of maintenance DNA methylation. Wehave recently identified another mechanism cou-pled with DNA replication machinery for the re-cruitment of DNMT1 to replicating chromatin inwhich UHRF1-mediated dual mono-ubiquitylationof PCNA-associated factor 15 (PAF15) plays an es-sential role. In Xenopus cell- free extracts, PAF15 ac-cumulates on chromatin with dual mono-ubiq-uitylation (PAF15Ub2) in a PCNA-, UHRF1- andDNA replication-dependent manner. PAF15Ub2 in-teracts with DNMT1 with a structural mode similarto that of H3Ub2. Suppression of DNMT1 interac-tion with H3Ub2 in the extract shows that PAF15and histone H3 have non-redundant roles in the re-cruitment of DNMT1 and subsequent DNA methyl-ation. Consistent with this, in mammals, PAF15 isalso subjected to UHRF1-dependent dual mono-ubiquitylation and is capable of binding to DNMT1.Mouse embryonic stem cells expressing PAF15 car-rying substitutions of lysine to arginine at ubiq-uitylation sites demonstrate a marked reduction inthe DNA methylation level. Together, our study re-veals that PAF15 and histone H3 have non-redun-dant roles in the regulation of DNA methylationmaintenance.

4. Regulatory mechanisms of chromosome seg-regation by mitotic rounding

Kotaro Nishimura1, Yoshikazu Johmura, KishoYokote, Yoshie Chiba, Toru Hirota2, Keiko Kono3

and Makoto Nakanishi: 1Memorial Sloan KetteringCancer Center, University of Copenhagen, 2CancerInstitute of the Japanese Foundation for CancerResearch, 3OIST

During mitosis, animal cells undergo dynamic re-organization of cell shape, from flat to round. Togenerate force for mitotic rounding, cells increasetheir cortical tension and intracellular pressure. Mi-totic cell rounding is critical for chromosome segre-gation, development, tissue organization, and tu-mor-suppression. Mitotic cell rounding requires atleast three key modules: 1) F-actin regulated byRhoA and an actin nucleator formin DIAPH1, 2)Myosin II regulated by Rac1 and Cdc42, and 3) the

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Ezrin, Radixin and Moesin (ERM) family proteins.DIAPH1 is a member of actin nucleator formin

family proteins, whose mutations are associatedwith various diseases including nonsyndromicdeafness and microcephaly. Formin family proteinsare defined by the formin homology 1 (FH1) andformin homology 2 (FH2) domains. The formin ho-mology 1 (FH1) domain is required for the interac-tion to the actin monomer-binding protein profilin,whereas FH2 domain is responsible for actin fila-ment nucleation. Diaphanous - related formins(DRFs) compose a subgroup activated by the bind-

ing of Rho-type small GTPases. DRFs are involvedin organizing various cytoskeletal structures suchas filopodia, lamellipodia and cytokinetic contrac-tile rings. Among them, DIAPH1 is required for ac-tin stress fiber formation and maintenance of corti-cal force during mitotic cell rounding. Therefore,we hypothesized that RhoA-DIAPH1-PFN1 axiscould be minutely regulated during mitosis. We arenow investigating mechanisms of how Cdk1, amaster regulator of mitosis, regulates this axis andcoordinates between mitotic rounding and chromo-some segregation.

Publications

1. Johmura, Y., Maeda, I., Suzuki, N., Wu, W.,Goda, A., Morita, M., Yamaguchi, K., Yamamoto,M., Nagasawa, S., Kojima, Y., Tsugawa, K.,Inoue, N., Miyoshi, Y., Osako, T., Akiyama, F.,Maruyama, R., Inoue, J-I., Furukawa, Y., Ohta,T., and Nakanishi, M. Fbxo22-mediated KDM4Bdegradation determines selective estrogen recep-tor modulator activity in breast cancer. J Clin In-vest, 128: 5603-5619, 2018.

2. Nakanishi, K., Niida, H., Tabata, H., Hori, Y.,Hattori, M., Johmura, Y., Yamada, C., Ueda, T.,

Takeuchi, K., Yamada, K., Nagata, KI, Waka-matsu, N., Kishi, M., Pan, Y.A., Ugawa, S., Shi-mada, S., Sanes, J.R., Higashi, Y., and Nakanishi,M. Isozyme-specific role of SAD-A in neuronalmigration during development of cerebral cortex.Cereb Cortex, doi: 10.1093/cercor/bhy253. 2018.

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