1. The biological functions of cell adhesion inhuman oncogenesis

Takeshi Ito, Yumi Tsuboi, Yuki Kumagai, MasaruKasai, Atsuko Nakamura, Toko Funaki, YutaroShiraishi, Yota Mitobe, Tomoko Masuda, HiromiIchihara, Kaoru Kiguchi, Motoi Oba,1 DaisukeMatsubara and Yoshinori Murakami: 1ResearchInstitute of Molecular and Cell 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(IgCAMs) and acts as a tumor suppressor in vari-ous cancers. By contrast, CADM1 rather promotescell invasion and metastasis in adult T-cell leuke-mia (ATL) or small cell lung cancer (SCLC). In or-der to elucidate molecular pathways involved inthis CADM1-mediated dual functions in oncogene-sis, mass spectrometry (MS) analysis was per-formed to identify a series of proteins associatedwith CADM1 in epithelial cells and SCLC. In epi-thelial cells, we have identified several molecules inthe tyrosine kinase pathways and found that inter-

action of CADM1 with these tyrosine kinases couldmodify the growth-associated signaling triggeredby tyrosine kinases. In SCLC, MS analysis identi-fied a series of proteins also important for humantumorigenesis. Molecular biological analyses, in-cluding shedding of CADM1 protein, was also in-vestigated in collaboration with others (1).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).

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 of

Department of Cancer Biology

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

Professor Yoshinori Murakami, M.D., Ph.D.Assistant Professor Takeharu Sakamoto, Ph.D.Assistant Professor Takeshi Ito, Ph.D.

教 授 医学博士 村 上 善 則助 教 博士（医学） 坂 本 毅 治助 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 adhesion in cancerinvasion and metastasis. Genomic and epigenomic abnormalities involved in hu-man tumors, including adult T-cell leukemia, cholangiocarcinoma, lung, breast,head and neck and urological cancers, are also being investigated.

33

Tokyo, 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-ment 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.These antibodies would be also promising to gen-

erate several therapeutic approaches, including ra-dioisotope-conjugated antibodies and chimeric anti-gen receptor-T cell therapy. One of the monoclonalantibodies established in this laboratory was usedto generate a novel radioisotope-conjugated anti-body for possible molecular targeting radio-therapyagainst a subset of cancer in collaboration with oth-ers using the metal chelator DOTA or NOTA and[67Cu], a b－-emitting radionuclide (2).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. The PIK3CA mutations weredetected in 13 (48％) of 27 primary tumors. The in-cidence or patterns of these mutations are inde-pendent of any specific clinico-pathological charac-teristics of tumors. When ctDNA was examined, 4(33％) of 12 cases carrying the mutated PIK3CAshowed the identical mutation in pre-surgeryplasma. Furthermore, 2 (50％) of 4 cases with mu-tated PIK3CA in pre-surgical plasma showed theidentical mutations in post-surgery plasma. Ourstudy suggest that ctDNA would provide a noveltool to predict the outcome of breast cancer even inits early stage (3).To unveil additional molecular mechanisms un-

derlying multistage carcinogenesis, genomic, epi-genomic, and transcriptional alterations in keymolecules in human tumorigenesis were examinedin various cancers in collaboration with others (4,13).

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

Yoshinori Murakami, Daisuke Matsubara, ZenichiTanei2, Makoto Hirata5, Koichiro Yuji6 and KoichiMatsuda5: 5Laboratory of Genome Technology and6Project Division of International Advanced Medi-cal Research, The Institute of Medical Science,The University of Tokyo

A large number of genomic DNA from normal

peripheral lymphocytes as well as serum samplesfrom more than 260, 000 cases with 51 diseases wascollected and preserved in BioBank Japan in the In-stitute of Medical Science, the University of Tokyo.These samples and information were shown to bevaluable to obtain the precise view of clinical fea-tures and genetic polymorphisms associated withthe onset of human diseases in collaboration with alarge study group of order-made medicine in Japan(5-7, 14).

4. Analyses of novel signalling pathways in can-cer cells, macrophages, and cancer-associatefibroblasts that are associated with tumorgrowth and metastasis.

Hiroki J. Nakaoka, Tetsuro Hayashi, Zen-ichi Ta-nei, Akane Kanamori, Toshiro Hara7, KeiichiroTada4, Masashi Fukayama2, Motoharu Seiki8,Yoshinori Murakami, and Takeharu Sakamoto:7Division of Cancer Cell Research, Institute ofMedical Science, The University of Tokyo, 8Facultyof Medicine, Institute of Medical, Pharmaceuticaland Health Sciences, Kanazawa University

Unlike most cells, cancer cells activate hypoxiainducible factor-1 (HIF-1) to use glycolysis even atnormal oxygen levels, or normoxia. Therefore,HIF-1 is an attractive target in cancer therapy.However, the regulation of HIF-1 during normoxiais not well characterised. We have recently demon-strated that Mint3 activates HIF-1 in cancer cellsand macrophages by suppressing the HIF-1 inhibi-tor, factor inhibiting HIF-1 (FIH-1) (8). We have re-vealed that Mint3-deficient mice show reduced me-tastasis, with no apparent effect on primary tumorgrowth. Mint3 deficiency in inflammatory mono-cytes, which strongly express the chemokine recep-tor CCR2 and are recruited toward chemokineCCL2 from metastatic sites, hampers glycolysis-de-pendent chemotaxis of cells toward metastatic sitesand inhibits VEGFA expression, similar to the ef-fects observed with HIF-1 deficiency. Host Mint3induces VEGFA-mediated E-selectin expression inthe endothelial cells of target organs, thereby pro-moting extravasation of cancer cells and microme-tastasis formation. Administration of E-selectin-neu-tralizing antibody also abolished host Mint3-medi-ated metastatic formation. Thus, targeting APBA3 isuseful for controlling metastatic niche formation byinflammatory monocytes (9).Subsequently, we have examined whether Mint3

depletion affects tumor malignancy in MMTV-PyMT breast cancer model mice. In MMTV-PyMTmice, Mint3 depletion did not affect tumor onsetand tumor growth, but attenuated lung metastases.Experimental lung metastasis of breast cancer Met-1cells derived from MMTV-PyMT mice also de-creased in Mint3-depleted mice, indicating that host

34

Mint3 expression affected lung metastasis ofMMTV-PyMT-derived breast cancer cells. Furtherbone marrow transplant experiments revealed thatMint3 in bone marrow-derived cells promoted lungmetastasis in MMTV-PyMT mice. Thus, targetingMint3 in bone marrow-derived cells might be agood strategy for preventing metastasis and im-proving the prognosis of breast cancer patients (10).Furthermore, we examined whether Mint3 in fi-

broblasts contributes to tumor growth. Mint3 deple-tion in mouse embryonic fibroblasts (MEFs) de-creased tumor growth of co-injected human breastcancer cells, MDA-MB-231 and epidermoid carci-noma A431 cells in mice. In MEFs, Mint3 also pro-moted cancer cell proliferation in vitro in a cell-cellcontact-dependent manner. Mint3-mediated cancercell proliferation depended on HIF-1, and furthergene expression analysis revealed that the cell ad-hesion molecule, L1 cell adhesion molecule(L1CAM), was induced by Mint3 and HIF-1 in fi-

broblasts. Mint3-mediated L1CAM expression in fi-broblasts stimulated the ERK signalling pathwayvia integrin a5b1 in cancer cells, and promoted can-cer cell proliferation in vitro and tumour growth. Incancer-associated fibroblasts (CAFs), knockdown ofMT1-MMP, which promotes Mint3-mediated HIF-1activation, or Mint3 decreased L1CAM expression.As MEFs, CAFs also promoted cancer cell prolifera-tion in vitro, and tumour growth via Mint3 andL1CAM. In human breast cancer specimens, thenumber of fibroblasts expressing L1CAM, Mint3and MT1-MMP was higher in cancer regions thanin adjacent benign regions. In addition, more phos-pho-ERK1/2-positive cancer cells existed in the pe-ripheral region surrounded by the stroma than inthe central region of solid breast cancer nest. Thus,Mint3 in fibroblasts might be a good target for can-cer therapy by regulating cancer cell-stromal cellcommunication. (11)

Publications

1. Shirakabe K, Omura T, Shibagaki Y, Mihara E,Homma K, Kato Y, Yoshimura A, Murakami Y,Takagi J, Hattori S, and Ogawa Y. Sheddingsusceptibility is determined at both post-tran-scriptional and post-translational levels. Sci Re-ports, 7:46174, 2017. (doi: 10.1038/srep46174.)

2. Fujiki K, Yano S, Ito T, Kumagai Y, MurakamiY, Kamigaito O, Haba H, and Tanaka K. AOne - Pot Three - Component Double - ClickMethod for Synthesis of [67Cu]-Labeled Bio-molecular Radiotherapeutics. Sci Reports, 7(1):1912, 2017. (doi: 10.1038/s41598-017-02123-2.)

3. Sato A, Tanabe M, Tsuboi Y, Ikemura M, TadaK, Seto Y, Murakami Y. Detection of thePIK3CA mutation in circulating tumor DNA asa possible predictive indicator for poor progno-sis of early-stage breast cancer. Journal of CancerTherapy, 9, 42-54, 2018. (DOI: 10.4236/jct.2018.91006)

4. Nakano T, Ito A, Matsubara D, Murakami Y,Fukayama M, Niki T. Tomoyuki Nakano1,2, Ka-nai Y, Amano Y, Yoshimoto T, Matsubara D,Shibano T, Tamura T, Oguni S, Katashiba S, ItoT, Murakami Y, Fukayama M, Murakami T,Endo S, and Niki T. Establishment of HighlyMetastatic KRAS Mutant Lung Cancer Cell Sub-lines in Long-term Three-dimensional Low At-tachment Cultures, PLoS One, 12(8):e0181342,2017. (doi: 10.1371/journal.pone.0181342.).

5. Hirata M, Nagai A, Kamatani K, Ninomiya T,Tamakoshi A, Yamagata Z, Kubo M, Muto K,Kiyohara Y, Mushiroda T, Murakami Y, Yuji K,Furukawa F, Zembutus H, Tanaka H, OhnishiY, Nakamura Y, BioBank Japan CooperativeHospital Group, Matsuda K. Overview of Bio-

Bank Japan Follow- up Data in 32 Diseases. JEpidemiology, 27(1), 22-28, 2017. (doi: 10.1016/j.je.2016.12.006.)

6. Hirata M, Kamatani Y, Nagai A, Kiyohara Y,Ninomiya T, Tamakoshi A, Yamagata Z, KuboM, Muto K, Mushiroda T, Murakami Y, Yuji K,Furukawa Y, Zembutsu H, Tanaka H, OhnishiY, Nakamura Y, BioBank Japan CooperativeHospital Group, Matsuda K. Cross-sectionalanalysis of BioBank Japan Clinical Data: ALarge Cohort of 200,000 Patients with 47 Com-mon Diseases. J Epidemiology, 27(1), 9-21, 2017.(doi: 10.1016/j.je.2016.12.003.)

7. Nagai A, Hirata M, Kamatani Y, Muto K, Ma-tsuda K, Kiyohara Y, Ninomiya T, TamakoshiA, Yamagata Z, Mushiroda T, Murakami Y,Yuji K, Furukawa Y, Zembutsu H, Tanaka T,Ohnishi Y, Nakamura Y, BioBank Japan Coop-erative Hospital Group, Kubo M. Overview ofthe BioBank Japan Project: Study Design andProfile. J Epidemiology, 27(1):2-8, 2017. (doi:10.1016/j.je.2016.12.005.)

8. Sakamoto T, Seiki M. Integrated functions ofmembrane-type 1 matrix metalloproteinase inregulating cancer malignancy: Beyond a prote-inase. Cancer Sci, 108, 1095-1100, 2017. (doi:10.1111/cas.13231.)

9. Hara T, Murakami Y, Seiki M, and Sakamoto T.Mint3 in bone marrow-derived cells promoteslung metastasis in breast cancer model mice.Biochem Biophys Res Commun, in press. 490(3),688-692, 2017. (doi: 10.1016/j.bbrc.2017.06.102.)

10. Hara T, Nakaoka HJ, Hayashi T, Mimura K,Hoshino D, Inoue M, Nagamura F, MurakamiY, Seiki M, Sakamoto T. Control of metastatic

35

niche formation by targeting APBA3/Mint3 ininflammatory monocytes. Proc Natl Acad SciU S A, 30, 114(22), E4416-E4424, 2017.

11. Nakaoka HJ, Tanei Z, Hara T, Weng JS, Kana-mori A, Hayashi T, Sato H, Orimo A, Otsuji K,Tada K, Morikawa T, Sasaki T, Fukayama M,Seiki M, Murakami Y, Sakamoto T. Mint3-medi-ated L1CAM expression in fibroblasts promotescancer cell proliferation via integrin a5b1 andtumour growth. Oncogenesis, 6(5), e334, 2017.(doi: 10.1038/oncsis.2017.27.)

12. Saitoh Y, Ohno N, Yamauchi J, Sakamoto T,Terada N. Deficiency of a membrane skeletalprotein, 4.1G, results in myelin abnormalities inthe peripheral nervous system. Histochem CellBiol, 148, 597-606, 2017. (doi: 10.1007/s00418-

017-1600-6.)13. Saito M, Goto A, Abe N, Saito K, Maeda D, Oh-

take T, Murakami Y, Takenoshita S. Decreasedexpression of CADMI and CADM4 are associ-ated with advanced stage breast cancer. Oncol-ogy Letters, 15: 2401-2406, 2018. (doi: 10.3892/ol.2017.7536)

14. Tanikawa C, Kamatani Y, Takahashi A, Momo-zawa Y, Leveque K, Nagayama S, Mimori K,Mori M, Ishii H, Inazawa J, Fuse N, Takai-Iga-rashi T, Shimizu A, Sasaki M, Yamaji T,Sawada N, Iwasaki M, Tsugane S, Naito M,Hishida A, Wakai K, Furusyo N, Yuji K, Mu-rakami Y, Kubo M, Matsuda K. GWAS identi-fies two novel colorectal cancer loci at 16q24.1and 20q13.12. Carcinogenesis, in press.

36

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

Takao Seki, Fumiki Iwanami, Yuko Hata1,Masaaki Oyama1, Jin Gohda2, Toshifumi Aki-zawa3, Taishin Akiyama and Jun-ichiro Inoue:1Medical Proteomics Laboratory, and 2Center forAsian Infectious Diseases, IMSUT; 3Setsunan Uni-versity.

Transcription factor NF-kB binds specifically to adecameric motif of nucleotide, kB site, and activates

transcription. The activation of NF-kB has beendemonstrated to be carried out post-translationallyupon 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 subsequently

Department of Cancer Biology

Division of Cellular and Molecular Biology分子発癌分野

Professor Jun-ichiro Inoue, Ph.D.Associate Professor Taishin Akiyama, Ph.D.Assistant Professor Yuu Taguchi, Ph.D.Assistant Professor Yuri Shibata, Ph.D.Assistant Professor Mizuki Yamamoto, Ph.D.

教 授 薬学博士 井 上 純一郎准教授 博士（薬学） 秋 山 泰 身助 教 博士（医学） 田 口 祐助 教 博士（生命科学） 柴 田 佑 里助 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).

37

translocates to the nucleus to activate transcriptionof target genes. This project is to identify moleculesthat 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 local-ization as RelB expression and activation.RelbVenus/Venus DC analyses revealed that DCs consist ofRelB－, RelBlow and RelBhigh populations. The RelBhigh

population, which included mature DCs with pro-jections, displayed RelB nuclear localization,whereas RelB in the RelBlow population was in thecytoplasm. Although both the RelBlow and RelB－populations barely showed projections, MHC II andco-stimulatory molecule expression were higher inthe RelBlow than in the RelB－ splenic conventionalDCs. Taken together, our results identify the RelBlow

population as a possible novel intermediate matura-tion stage of cDCs and the RelbVenus/Venus mice as auseful tool to analyze the dynamic regulation of thenon-canonical NF-kB pathway.

2. HTLV-1 Tax induces formation of the activemacromolecular IKK complex by generatingLys63- and Met-1-linked hybrid polyubiquitinchains

Yuri Shibata, Jin Gohda2 and Jun-ichiro Inoue

Activation of NF-kB by human T-cell leukemiavirus type 1 (HTLV-1) Tax is thought to be crucial

in T-cell transformation and the onset of adult T-cell leukemia (ATL). Therefore, a better understand-ing of the precise mechanism underlying aberrantNF-kB activation is essential to develop new thera-peutic approaches. It is well known that Tax acti-vates NF-kB through activation of the IKK complexby generating Lys63-linked polyubiquitin chains.However, the molecular mechanism underlyingTax-induced IKK activation is not fully understood.In this study, we demonstrate that Tax recruits lin-ear (Met1-linked) ubiquitin chain assembly complex(LUBAC) to the IKK complex and that Tax fails toinduce IKK activation in cells that lack LUBAC ac-tivity. The ubiquitin absolute quantification(ubiquitin-AQUA) analyses revealed that bothLys63-linked and Met1-linked polyubiquitin chainsare associated with the IKK complex. Furthermore,treatment of the IKK-associated polyubiquitinchains with Met1-linked-chain-specific deubiquit-inase (OTULIN) resulted in the reduction of highmolecular weight polyubiquitin chains and the gen-eration of short Lys63-linked ubiquitin chains, indi-cating that Tax can induce the generation of Lys63-and Met1-linked hybrid polyubiquitin chains. Wealso demonstrate that Tax induces formation of theactive macromolecular IKK complex and that theblocking of Tax-induced polyubiquitin chain syn-thesis inhibited formation of the macromolecularcomplex. Taken together, Tax triggers Lys63- andMet1-linked hybrid polyubiquitin chains by recruit-ing LUBAC to the IKK complex, leading to the for-mation of the active macromolecular IKK complex.

3. Molecular mechanism of RANK signaling inosteoclastogenesis

Yuu Taguchi, Yo Yumiketa, Yui Iwamae, YuukiNakano, Mikako Suzuki, Yoko Hirayama, Masaa-ki Oyama1, Hiroko Kozuka-Hata1, Jin Gohda2, andJun-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-

38

tial for osteoclastogenesis. The RANK signal acti-vates transcriptional factors, NF-kB and AP-1,through the E3 ubiquitin ligase TRAF6, and also in-duces activation of PLCg2-mediated Ca2＋ signalingpathway. These signals lead to the induction ofNFATc1, a master transcriptional factor in osteo-clastogenesis. We have previously demonstratedthat RANK has a functional amino acid sequences,named Highly Conserved domain in RANK (HCR),which does not have any homology of amino-acidsequence with other proteins. The HCR acts as aplatform for formation of signal complex includingTRAF6, PLCg2 and adaptor protein Gab2. This for-mation of signal complex is involved in sustainingactivation of RANK signaling, and is essential forthe NFATc1 induction and osteoclastogenesis. Toelucidate other functions and the precise molecularmechanism of HCR, we have performed yeast two-hybrid screening and protein-array to identify theinteracting protein to receptor RANK includingHCR. Some candidate proteins were associatedwith RANK and HCR, and were involved in the in-duction of osteoclast-specific gene expression, sug-gesting that HCR has an additional function otherthan NFATc1 induction. We are currently investi-gating the molecular mechanisms of these candi-date proteins in osteoclastogenesis. Moreover, to re-veal the novel mechanisms involved in osteoclasto-genesis, we performed microarray analysis of geneexpression levels during osteoclastogenesis. Sincesome genes were dramatically downregulated in re-sponse to RANKL stimulation, we are currently in-vestigating whether these genes are involved in theregulation of osteoclastogenesis in vitro and in vivo.Furthermore, to identify the novel genes which areinvolved in osteoclastogenesis, we are now tryingto construct the screening system using CRISPR/Cas9 system. Moreover, we tried to elucidate theTRAF6-dependent molecular mechanism at the sub-sequent step of NFATc1 induction in osteoclasto-genesis such as cell-cell fusion and actin ring for-mation.

4. TRAF6 regulates pregnancy-induced mam-mary gland development and maintenance ofepithelial stem cells

Mizuki Yamamoto, Chiho Abe and Jun-ichiroInoue

Mammary gland development is characterized bythe unique process by which the epithelium in-vades the stroma. During puberty, tubule formationis coupled with branching morphogenesis which es-tablishes the basic arboreal network emanatingfrom the nipple. During pregnancy, the ductal cellsundergo rapid proliferation and form alveolarstructures within the branches for milk production.Upon weaning of the pups, lactation stops and the

mammary gland undergoes rapid involution.RANK signaling triggered by progesterone-in-

duced RANKL leads to mammary stem cell (MaSC)generation and promotes pregnancy-induced epi-thelial cell differentiation and expansion to enablemammary gland development. RANK activatesthree pathways, including the canonical and non-canonical NF-kB pathways and the pathway thatinduces Id2 nuclear translocation. While the Id2pathway leads to cell survival and maturation, thedistribution of roles played by the two NF-kB path-ways remains to be elucidated. To determine thefunction of TRAF6-canonical NF-kB pathway onmammary gland development, we are analyzingTRAF6-deficient mammary gland structure andgene expression profiles, and found that TRAF6-ca-nonical NF-kB pathway regulates pregnancy-in-duced epithelial cell expansion.

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

Mizuki Yamamoto, Aya Watanabe and Jun-ichiroInoue

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 choseHCC38, a human triple-negative breast cancer cellline, because HCC38 is composed of epithelial andmesenchymal populations at a fixed ratio eventhough mesenchymal cells proliferate significantlymore slowly than epithelial cells. We purified epi-thelial and mesenchymal cells from Venus-labeledand unlabeled HCC38 and mixed them at variousratios to follow EMT and MET. Using this system,we demonstrate that the efficiency of EMT is aboutan order of magnitude higher than that of MET andthat the two populations significantly enhance thetransition of cells from the other population to theirown. In addition, knockdown of ZEB1 or SLUG sig-nificantly suppressed EMT but promoted partialMET, indicating ZEB1 and SLUG are crucial toEMT and MET. We also demonstrate that primarybreast cancer cells underwent EMT that correlatedwith changes in expression profiles of genes deter-mining EMT status and breast cancer subtype.These changes were very similar to those observedin EMT in HCC38. Consequently, we proposeHCC38 as a suitable model to analyze EMT-MET

39

dynamics that could affect development of triple-negative breast cancer.

6. Long-term hindlimb unloading of mice causesa reduction of thymic epithelial cells express-ing autoimmune regulator

Riko Yoshinaga, Ryosuke Tateishi, Kenta Horie,Nobuko Akiyama, Jun-ichiro Inoue and TaishinAkiyama

Space flight impacts various physiological sys-tems of astronauts. For health management duringspace flight, understanding effect of space flight onthe physiological systems should be important.However, because space flight involves high oper-ating costs, several ground models of space flightwere developed. Hindlimb unloading (HU) of ro-dents has been used as a ground model of space-flight in terms of anti-orthostasis, stress, and inac-

tivity. Whereas effect of HU on mice for short-pe-riod were relatively characterized well, effect oflong-term HU on mice were not well-characterized.We have analyzed influences of 14-days HU onmurine thymus. Data showed that thymic mass andtotal thymic cellularity were reduced by the 14-days HU. In addition, plasma corticosterone levelwas slightly increased, suggesting a HU-dependentstress on mice. Although corticosterone reportedlycauses a decrement in CD4＋CD8＋ (DP) thymo-cytes, the reduction of thymocytes caused by longterm HU were not selective for DP thymocytes. Wefound that medullary thymic epithelial cells ex-pressing autoimmune regulator were reduced andthymic B cells were rather increased by long-termHU. Our data showed that HU influences both thy-mocytes and thymic cells required for self-tolerantT cell selection. which may disturb T cell-mediatedimmune responses.

Publications

Varney, M., Choi, K., Bolanos, L., Christie, S., Fang,J., Grimes, L., Maciejewski, J., Inoue, J. andStarczynowski D.＊ Epistasis between TIFAB andmiR-146a, neighboring genes in del(5q) MDS.Leukemia 31, 491-495 (2017). doi:10.1038/leu.2016.276.

Shibata, Y., Tokunaga, F., Goto, E., Komatsu, G.,Gohda, J., Saeki, Y., Tanaka, K., Takahashi, H.,Sawasaki, T., Inoue, S., Oshiumi, H., Seya, T.,Nakano, H., Tanaka, Y., Iwai, K. and Inoue, J.HTLV-1 Tax Induces Formation of the ActiveMacromolecular IKK Complex by GeneratingLys63- and Met1-Linked Hybrid PolyubiquitinChains. PLoS Pathogens 13(1):e1006162 (2017).doi: 10.1371/journal.ppat.1006162.

Into, T.,＊ Horie, T., Inomata1, M., Gohda, J., Inoue,J., Murakami, Y. and Niida., S. Basal autophagyprevents autoactivation or enhancement of in-flammatory signals by targeting monomericMyD88. Sci. Rep. 7: 1009 (2017). doi:10.1038/s41598-017-01246-w.

Yamamoto, M., Sakane, K., Tominaga, K., Gotoh,N., Niwa, T., Kikuchi, Y., Tada, K., Goshima, N.,Semba, K. and Inoue, J.＊ Intratumoral bidirec-

tional transitions between epithelial and mesen-chymal cells in triple-negative breast cancer. Can-cer Sci. 108, 1210-1222 (2017). doi: 10.1111/cas.13246.

Inuki, S., Aiba, T., Kawakami,S., Akiyama, T.,Inoue, J. and Fujimoto, Y.＊ Total synthesis of D-glycero-D-manno-Heptose 1,7-Bisphosphate andevaluation of its ability to modulate NF-kB Acti-vation. Org. Lett. 19, 3079-3082 (2017). doi:10.1021/acs.orglett.7b01158.

Magilnick, N., Reyes, E.Y., Wang, W-L., Vonder-fecht, S.L., Gohda, J., Inoue, J. and Boldin, M.P.＊The miR-146a-Traf6 regulatory axis controls auto-immunity and myelopoiesis, but is dispensablefor hematopoietic stem cell homeostasis and tu-mor suppression. Proc. Natl. Acad. Sci. USA 114,E7140-E7149 (2017). doi:10.1073/pnas.1706833114

Koga, R., Radwan, M.O., Ejima, T., Kanemaru, Y.,Tateishi, H., Ali, T.F.C., Ciftci, H.I., Shibata, Y.,Taguchi, Y., Inoue, J., Otsuka M. and Fujita, M.＊A Dithiol Compound Binds to the Zinc FingerProtein TRAF6 and Suppresses its Ubiquitination.ChemMedChem 12, 1935-1941 (2017). doi:10.1002/cmdc.201700399.

40

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

Ueta, R., Eguchi, T., Tezuka, T., Izawa, Y., Mi-yoshi, S., Weatherbee, SD.1, Nagatoishi, S.2, Tsu-moto, K.2, and Yamanashi, Y.: 1Department of Ge-netics, Yale University. 2Medical Proteomics Labo-ratory, IMSUT.

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, as

described 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.

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

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.

41

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 that Dok-7 directly interacts with the cytoplasmic portion ofMuSK and activates the RTK, and that neural agrinrequires Dok-7 in order to activate MuSK. Indeed,in vivo overexpression of Dok-7 increased MuSKactivation and promoted NMJ formation. Con-versely, mice lacking Dok-7 formed neither NMJsnor AChR clusters. In addition, we have recentlyfound that postnatal knockdown of dok-7 gene ex-pression in mice causes structural defects in NMJsand myasthenic pathology, suggesting an essentialrole for Dok-7 not only in the embryonic formationbut also in the postnatal maintenance of NMJs.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-7

knockout 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.We are investigating how the C-terminal regionacts 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 molecular mechanisms under-lying the agrin-mediated postnatal maintenance ofNMJs.

3. Pathophysiological mechanisms underlyingDOK7 myasthenia.

Tezuka, T., Eguchi, T., Saito A., Ueta R., Ari-mura, S., Fukudome T.1, Sagara H.2, Motomura,M.3, Yoshida, N.4, Beeson, D.5, and Yamanashi, Y.:1Department of Neurology, Nagasaki KawatanaMedical Center. 2Medical Proteomics Laboratory,IMSUT. 3Department of Engineering, Faculty ofEngineering, Nagasaki Institute of Applied Sci-ence. 4Laboratory of Developmental Genetics, IM-SUT. 5Weatherall Institute of Molecular Medicine,University of Oxford.

As mentioned above, impaired clustering of

42

AChRs 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.

4. DOK7 gene therapy that enlarges NMJs.

Arimura, S., Miyoshi, S., Ueta, R., Kanno, T., Te-zuka, T., Okada, H.1, Kasahara, Y.1, Tomono, T.1,Motomura, M.2, Yoshida, N.3, Beeson, D.4, Takeda,S.5, Okada, T.1, and Yamanashi, Y.: 1Departmentof Biochemistry 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 de-creased 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 indetail.

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 AChRantibody-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 AChR

43

antibody-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; Exp. Neurol., 297: 158-167, 2017).Given that Lrp4 antibodies are found in patients ofamyotrophic lateral sclerosis (ALS) (Ann. Clin.Transl. Neurol. , 1:80-87, 2014), which is associatedwith NMJ defects, Lrp4 antibodies may be involvedin a range of neuromuscular disorders that featuredefects in NMJs, including those of unknown etiol-ogy. We are investigating pathogenicity of Lrp4 an-tibodies.

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

Arimura, S., Kajikawa, S., Kanno, T., Jozawa, H.,Yamazaki, S., 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 Yama-nashi, Y.: 1Laboratory of Systems Biology, IM-SUT. 2Department of Biological Signaling andRegulation, Faculty of Medicine, University ofTsukuba. 3Department of Skeletal Molecular Phar-macology, Medical Research Institute, TokyoMedical and Dental University. 4Division of Cellu-lar and Molecular Biology, IMSUT. 5Departmentof Pathology, Tokyo Women's Medical University.6Laboratory of Developmental Genetics, 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 key

negative 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. In addition, we have recently foundthat Dok-1 and Dok-2 negatively regulate intestinalinflammation in the dextran sulfate sodium-in-duced colitis model, apparently through the induc-tion of IL-17A and IL-22 expression. We are furtherinvestigating roles of Dok-1 to Dok-6, includingthose in tumor malignancy, inflammatory disor-ders, bone homeostasis, and other types of intracta-ble diseases.

7. Omic analyses.

Ueta, R., Tezuka, T., Arimura, S., Eguchi, T.,Jozawa, H., Saito A., Miyoshi. S., Takada Y.1,Iemura, S.2, Natsume, T.3, Kozuka-Hata, H.4,Oyama, M.4, and Yamanashi, Y.: 1Laboratory ofGlyco-bioengineering, The Noguchi Institute,2Translational Research Center, Fukushima Medi-cal University. 3National Institute of Advanced Sci-ence and Technology, Molecular Profiling Re-search Center for Drug Discovery. 4Medical Pro-teomics 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 Mesdc2also 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 glycosylation

44

and 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 atrophy.

8. Screening of chemical compound and siRNAlibraries.

Ueta, R., Kosuge, M., Yamazaki, S., Nagatoishi,S.1, Tsumoto, K.1, and Yamanashi, Y.: 1MedicalProteomics Laboratory, 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.

Publications

Miyoshi S., Tezuka T., Arimura S., Tomono T.,Okada T., and Yamanashi Y. DOK7 gene therapyenhances motor activity and life span in ALSmodel mice. EMBO Molecular Medicine, 9: 880-889(2017)

Yamasaki T., Deki-Arima N., Kaneko A., MiyamuraN., Iwatsuki M., Matsuoka M., Fujimori-TonouN., Okamoto-Uchida Y., Hirayama J., Marth J.D.,Yamanashi Y., Kawasaki H., Yamanaka K., Pen-ninger J.J., Shibata S., and Nishina H. Age-de-

pendent motor dysfunction due to neuron-spe-cific disruption of stress-activated protein kinaseMKK7. Scientific Reports 7: 7348 (2017)

Ueta R., Tezuka T., Izawa Y., Miyoshi S., Naga-toishi S., Tsumoto K., and Yamanashi Y. The car-boxyl-terminal region of Dok-7 plays a key, butnot essential, role in activation of muscle-specificreceptor kinase MuSK and neuromuscular syn-apse formation. J. Biochemistry , 161: 269-277(2017)

45

1. Regulatory mechanisms of aging and carcino-genesis by accumulation of senescent cellsin vivo

Yoshikazu Johmura, Chieko Konishi, SayakaYamane, Yoshie Chiba, Dan Li, Kazuhiro Hitomi,Takehiro Yamanaka, Kisho Yokote, NarumiSuzuki, Shizuka Takeyama, Honoka Hagiwara,and Makoto Nakanishi:

One important hallmark of senescence is the in-ability to proliferate in response to physiologicalmitotic stimuli. The limited lifespan of human cellsis governed by telomere shortening as well as vari-ous genotoxic stressors, all of which ultimately acti-vate DNA damage responses. We and others haverecently uncovered the molecular mechanisms in-volved in permanent cell cycle arrest during the se-nescence process in which p53 activation at G2plays a necessary and sufficient role by inducing amitosis skip. Another hallmark of senescence is theappearance of senescence-associated secretory phe-notypes (SASP), such as robust secretion of numer-ous growth factors, cytokines, proteases, and otherproteins, that can cause deleterious effects on the

tissue microenvironment. On the other hand, SASPalso has positive effects on the repair of damagedtissue, at least at a young age. Induction of thesetwo hallmarks of senescence is often coordinated,but their respective mechanisms do not alwaysoverlap. Most notably, p38MAPK is critically re-quired for SASP through activating NF-kB inde-pendently of canonical DDR, but p53 restrainsp38MAPK, leading to the suppression of SASP insenescent cells. There appear to be missing linksthat could more fully explain the antagonistic ef-fects of p53 on the induction of these two represen-tative hallmarks of senescence.The key to the regulation of p53 activity is con-

trol of the stability of its protein, which is mainlyorchestrated through a network of ubiquitylationreactions, although other mechanisms such as regu-lation of its localization are also involved. Whilenumerous E3 ubiquitin ligases for p53 have beenreported, data are less clear regarding the in vivorelevance of these E3 ligases in p53 regulation ex-cept for murine double minute 2 (Mdm2) Mdm2 isitself a transcriptional target of p53, and acts to cre-ate a negative feedback loop. Importantly, in micewith a disrupted p53-Mdm2 feedback loop, the

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.Senior Assistant Professor Atsuya Nishiyama, Ph.D.Assistant Professor Yoshikazu Johmura, Ph.D.

教 授 医学博士 中 西 真講 師 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.

46

degradation profile of p53 upon DNA damage ap-peared to be normal, calling the role of Mdm2 asthe sole E3 ubiquitin ligase for stress-induced p53into question. In order to uncover the mechanismsunderlying negative regulation of p53 during senes-cence maintenance, we performed gene expressionanalysis using normal and sorted senescent cellsand found that Fbxo22 was highly expressed in se-nescent cells in a p53-dependent manner. Moreover,SCFFbxo22 ubiquitylated p53 and formed a complexwith a lysine demethylase, KDM4A. Ectopic expres-sion of a catalytic mutant of KDM4A stabilized p53and enhanced p53 interaction with PHF20 in thepresence of Fbxo22. SCFFbxo22-KDM4A was requiredfor the induction of p16 and senescence-associatedsecretory phenotypes at the late phase of senes-cence. Fbxo22－/－ mice were almost half the size ofFbxo22＋/－ mice due to the accumulation of p53.These results indicate that SCFFbxo22-KDM4A is an E3ubiquitin ligase that targets methylated p53 andregulates key senescent processes.

2. Regulation of maintenance DNA methylationby two-mono ubiquitylated substrates

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

Methylation of the cytosine residue at CpG siteshas a crucial role in early embryonic developmentand cellular differentiation in vertebrates. Mainte-nance DNA methylation is mainly regulated byDnmt1, which converts hemi-methylated DNA toits fully methylated form. We have recently unrav-eled a mechanism underlying Dnmt1 recruitment tohemi-methylated DNA sites by Uhrf1 (Ubiquitin-like, containing PHD and RING finger domains 1)in which Uhrf1-dependent ubiquitylation of histoneH3 (H3) plays an essential role. Recently, ubiquitininteracting motif (UIM) within replication foci tar-geting sequence domain (RFTS) of Dnmt1 was pro-posed as an ubiquitylated H3 (H3Ub) binding re-gion. However, for the rigorous inheritance of DNAmethylation patterns, recognition of hemi-methyl-ated DNA region by Dnmt1 must be of high affin-ity and specific, suggesting the existence of aunique and unidentified module of H3Ub recogni-tion by Dnmt1.Fine-tuned regulation of DNA methyltransferase

activities is also required for rigorous inheritance ofDNA methylation patterns. Recently, unexpectedregulatory principles of DNA methyltransferases(DNMTs) were identified, in which their catalytic

activities are auto-inhibited by their intramoleculardomain-domain interactions. In the case of Dnmt1,crystal structure of nearly full-length Dnmt1 re-vealed that the RFTS is deeply inserted into theDNA-binding pocket of the catalytic domain, indi-cating its auto-inhibitory mode. Thus, above twodistinct functions of RFTS suggest the molecularcoupling between targeting and activation ofDnmt1 at DNA replication sites.To address this important issue, we identified

two mono ubiquitylated histone H3 as a uniqueand specific structure that is preferentially recog-nized by Dnmt1. In addition, crystal structure ofRFTS of Dnmt1 in complex with H3-K18/23Ub2 re-vealed that the two ubiquitins were simultaneouslybound to RFTS via canonical hydrophobic andatypical hydrophilic interactions. The C-lobe ofRFTS together with K23Ub surface also recognizedN-terminal tail of H3. Binding of H3-K18/23Ub2also underwent spatial rearrangement of two lobesin RFTS, suggesting the opening of its active site.Incubation of Dnmt1 with H3-K18/23Ub2 drasti-cally increased its catalytic activity in vitro. Our re-sults thus shed light on the essential role of previ-ously unidentified and unique module of Dnmt1,which recognizes H3Ub2 in rigorous maintenanceof DNA methylation.

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

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

and Makoto Nakanishi: 1BRIC, University of Co-penhagen, 2Cancer Institute of the Japanese Foun-dation for Cancer Research, 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) theEzrin, 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-

47

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. Ishiyama, S., Nishiyama, A., Saeki, Y., Moritsu-gu, K., Morimoto, D., Yamaguchi, L., Arai, N.,Matsumura, R., Kawakami, T., Mishima, Y.,Hojo, H., Shimamura, S., Ishikawa, F., Tajima, S.,Tanaka, K., Ariyoshi, M., Shirakawa, M.,Ikeguchi, M., Kidera, A., Suetake, I., Arita, K.,and Nakanishi, M. Structure of the Dnmt1 readermodule complexed with a unique two-mono-ubiquitin mark on histone H3 reveals the basisfor DNA methylation maintenance. Mol Cell, 68:350-360, 2017.

2. Iwata, T., Uchino, T., Koyama, A., Johmura, Y.,Koyama, K., Saito, T., Ishiguro, S., Arikawa, T.,Komatsu, S., Miyachi, M., Sano, T., Nakanishi,M., and Shimada, M. The G2 checkpoint inhibi-tor CBP-93872 increases the sensitivity of colorec-tal and pancreatic cancer cells to chemotherapy.PLoS One, 12: e0178221, 2017.

3. Yamaguchi, L., Nishiyama, A., Misaki, T.,Johmura, Y., Ueda, J., Arita, K., Nagao, K.,Obuse, C., and Nakanishi, M. Usp7-dependenthistone H3 deubiquitylation regulates mainte-nance of DNA methylation. Sci Rep., 7: 55, 2017.

4. Negishi, Y., Miya, F., Hattori, A., Johmura, Y.,Nakagawa, M., Ando, N., Hori, I., Togawa, T.,Aoyama, K., Ohashi, K., Fukumura, S., Mizuno,S., Umemura, A., Kishimoto, Y., Okamoto, N.,Kato, M., Tsunoda, T., Yamasaki, M., Kanemura,Y., Kosaki, K., Nakanishi, M., and Saitoh, S. Acombination of genetic and biochemical analysesfor the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly. BMC Med Genet., 18:4, 2017.

5. 城村由和，中西真．細胞老化の誘導・維持におけるp５３の機能．実験医学．羊土社．３５（１４）２３４５―２３５０，２０１７．

48