Whatʼs New from Juntendo University, Tokyo Juntendo Medical Journal 2017. 63(5), 322-325

Peking University - Juntendo University Joint Symposium on Cancer Research and Treatment ADAM28 (a Disintegrin and 28) in Cancer Cell Proliferation and Progression

YASUNORI OKADA＊

＊Department of Pathophysiology for Locomotive and Neoplastic Diseases, Juntendo University Graduate School of Medicine, Tokyo, Japan

A disintegrinandmetalloproteinase 28 (ADAM28) is overexpressedpredominantlyby carcinoma cells in more than 70% of the non-small cell lung carcinomas, showing positive correlations with carcinoma cell proliferation and metastasis. ADAM28 cleaves insulin-like growth factor binding protein-3 (IGFBP-3) in the IGF-I/IGFBP-3 complex, leading to stimulation of cell proliferation by intact IGF-I released from the complex. ADAM28 also degrades von Willebrand factor (VWF), which induces apoptosis in human carcinoma cell lines with negligible ADAM28 expression, andthe VWF digestionby ADAM28-expressing carcinoma cells facilitates them to escape from VWF-induced apoptosis, resulting in promotion of metastasis. We have developed human antibodies against ADAM28 andshown that one of them significantly inhibits tumor growth andmetastasis using lung adenocarcinoma cells. Our data suggest that ADAM28 may be a new molecular target for therapy of the patients with ADAM28-expressing non-small cell lung carcinoma. Key words: a disintegrin and metalloproteinase 28 (ADAM28), cell proliferation, invasion, metastasis, human antibody inhibitor

Introduction human cancers 2). However, development of the synthetic inhibitors of MMPs andtheir application Cancer cell proliferation andprogression are for treatment of the cancer patients failed 3). modulated by proteolytic cleavage of tissue micro- On the other hand, members of the ADAM (a environmental factors such as extracellular matrix disintegrin and metalloproteinase) family, (ECM), growth factors andcytokines, receptors another family belonging to the metzincin gene andcell adhesionmolecules. Matrix metalloprotei- family, have recently attractedattention, since they nases (MMPs) in the metzincin gene family play an are implicatedin various pathophysiological events important role in the modulation through degrada- such as morphogenesis, inflammation andcancers by tion of ECM and/or non-ECM molecules in human proteolytic processing of transmembrane proteins, malignant tumors, leading to promotion of cancer cleavage of secretedfactors andmodulationof cell cell invasion andmetastasis 1)-3). When focusedon adhesion and signaling events 4)-6) (Table-1). ADAM carcinoma cell-derived MMPs, MMP-2 activated members are composed of common domains includ- by membrane-type 1 MMP andMMP-7 anchored ing propeptide, metalloproteinase, disintegrin-like, to cell membranes by CD151 are known to be cysteine-rich, epidermal growth factor (EGF)-like, important in cancer cell invasion andmetastases in transmembrane andcytoplasmic domains 5) 7). They

Yasunori Okada Department of Pathophysiology for Locomotive and Neoplastic Diseases, Juntendo University Graduate School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan TEL: +81-3-5800-7531 FAX: +81-3-5800-7532 E-mail: [email protected] 〔Received Dec. 24, 2016〕〔AcceptedJan. 23, 2017〕

Copyright © 2017 The Juntendo Medical Society. This is an open access article distributed under the terms of Creative Commons Attribution Li- cense (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original source is properly credited. doi: 10.14789/jmj.63.322

322 Juntendo Medical Journal 63(5), 2017 B ligand; IGFBP-3, κ Lymphatic system, gastrointestinal system system, pancreas, pituitary gland Testis Testis Testis Testis Testis Brain Testis thelial cell, osteoclast, synovial cells chondrocyte, placenta Various tissues Kidney, brain, chondrocyte Brain 1 Smooth muscle cell, chondrocyte, endo- 1, β β 9 9 ivator of nuclear facto α α 1, 1 Lung (flbroblast, smooth muscle). uterus 1 Testis, lung, lymphocyte, gstrointestinal 1 Osteoblast, muscle, 4, 1 Testis, erythrocyte 1 Testis β β β β β β β 5 9 9 9 6 9 9 kin-6 receptor; KL-1, Kit-ligand-1. α α α α α α α 1, 1, 7, 1 Various tissues 1, 1, 7, 1, β β β β β β β β 4 5 4 5 7 6 4 6 α α α α α α α α 3 Brain, heart 3, 5 1, 1, 1, 1 Macrophage, various tissues 1, 1, 1, 1, β β β β β β β β β β β 4 4 V 4 5 V V 4 2 4 4 α α α α α α α α α α α , α , RANKL Macrophage, neutrophil α , proTGF- , collagen IV, gelatin, α α APP, KL-1, insulin B chain CD23, von Willebrandfactor Proneuregulin, RANKL TNF-p75 receptor, ErbB4, TRANCE, proHB-EGF, proamphiregulin, proepiregulin, APP, IL6R, CD44, L-selectin ProTNF- Myelin basic protein, Delta,L1, APP, CD44, proHB-EGF, Notch, Delta-like 1, Jagged, N-cadherin, E-cadherin, VE-cadherin, Ephrin A2, Ephrin A5 FASL, IL6R ProHB-EGF, IGFBP-3 and5, proepiregulin collagen IV, gelatin, fibronectin CD23, proTNF APP, fibronectin, gelatin The human ADAM gene family 1 - Table Genetically relatedto bronchial asthma andcardiovascularorgans angiogenesis adipogenesis infiltration Potential functions Substrates Integrin-binding Sites of expression P, Secretedform Growth factor metabolism Myelin basic protein, IGFBP-3, NP P, Cytoplasmic form Arteriosclerosis, angiogenesis Collagen IV, gelatin PP, Secreted form Sheddase, development, Sheddase, myogenesis, P, Secreted form Shaddase, cell migration ProHB-EGF, TNF-p75 receptor, non-proteinase type (NP) andsplice variants (isoforms) NP Sperm/egg binding/fusion β , MCMP, , FKSG34 P, N-terminal form Sheddase, formation of neuron β α γ , Fertilin- β MDC-Ls tMDC III AD56, CR II-7 Kuzbanian MLTN, MLTNA Meltrin- ADAMDEC1 P ADAM29ADAM30 svph 1ADAM32 svph 4ADAM33 AJ131563 NP P NP P ADAM28 e-MDC II, MDC-Lm, ADAM21ADAM22 ADAM31ADAM23 MDC2 MDC3 P NP, Cytoplasmic form NP ADAM20 P Formation of sperm ADAM18 ADAM27, ADAM19 Meltrin- ADAM17 TACE, cSVP P Sheddase, heart development ProTNF- ADAM15 Metargidin, MDC15, ADAM10 MDAM, ADAM11ADAM12 MDC Meltrin- NP, Secretedform Tumor suppressor gene? ADAM8 MS2ADAM9 (CD156) MDC9, MCMP, P Shaddase, neutrophil ADAM7 EAP I, GP-83 NP ADAM Other namesADAM2 PH-30 Proteinase-type (P) or insulin-like growth factor binding protein-3; TGF, transforming growth factor; TRANCE, TNF-related activation induced cytokine; IL6R, interleu Abbreviations: HB-EGF, heparin-binding epidermal growth factor; APP, amyloid precursor protein; TNF, tumor necrosis factor; RANKL, receptor act

323 Okada: ADAM28 in cancer cell proliferation and progression

include 21 members, among which 13 members are serum levels of ADAM28, andfoundthat the level is proteinase-type ADAMs (Table-1). Many mem- significantly higher in the patients than in the brane proteins including growth factors such as EGF, control normal subjects 9). The levels were signifi- heparin-binding EGF, transforming growth factor-α cantly increasedwith advancesof tumor stage, and andcytokines such as tumor necrosis factor- α significantly higher in the patients with recurrent (TNF-α) are synthesizedas precursors andactivated carcinoma or lymph node metastasis. When the by processing with proteinases. In addition, a number proportion of ADAM28-immunostainedcarcinoma of cell surface receptors including TNF-α receptor-I, cells to total carcinoma cells (immunoreactivity) in TNF-α receptor-II, CD44, L-selectin andErb4/HER4 the adenocarcinomas with a tumor size of ≤20 mm undergo cleavage near the transmembrane domain, a in diameter was compared with the 7-year survival process called ectodomain shedding 4). In both cases, of the patients, disease-free and overall survivals ADAMs play a major role. ADAM members have cell were significantly lower in the ADAM28 high- adhesion properties by interaction with integrins and expressing group than in the low-expressing other proteins such as syndecans and fibronectin, group 9). These data suggest that the serological which may be involvedin cancer cell motility, invasion and/or histological assessment of ADAM28 may be andmetastasis. Thus, ADAMs are expectedto play diagnostic and prognostic markers for non-small roles in cancer cell proliferation andprogression cell lung carcinoma patients. through proteolytic and/or non-proteolytic modula- To examine the regulation mechanisms of tion of membrane andsecretedproteins. ADAM28 gene expression, we investigatedthe expression of ADAM28 in Madin-Darby canine Expression of ADAM28 in human non-small cell kidney epithelial cells transformed by several lung carcinomas oncogenes, andfoundthat v-src transformants selectively induce ADAM28 10). We also showedthat To study the expression of ADAM members, the the ADAM28 expression in human carcinoma cell mRNA expression of proteinase-type ADAM lines is correlatedwith phosphorylation of Src, and species including ADAM9, 10, 12, 15, 17, 19, 20, 21, demonstrated co-expression of ADAM28 and 28 and30 was examinedin human non-small cell phosphorylatedSrc in neoplastic cells of the lung, lung carcinomas 8). RT-PCR showedthat among the breast andcolon carcinomas. In both v-src trans- ADAM species examined, only ADAM28, which is formants andhuman ADAM28-expressing carci- composedof prototype membrane-type ADAM28m noma cell lines, activation of Src andits downstream andshort secreted-typeADAM28s, appearedto be MEK/ERK andPI3K/mTOR signaling pathways selectively expressedin all the carcinoma tissues. were essential to overexpression of ADAM28 10). Real-time quantitative PCR indicated that the relative expression levels of ADAM28m and Substrates and functions of ADAM28 ADAM28s are significantly more than 10-fold in carcinoma cell proliferation and metastasis higher in the carcinomas than in the control non- neoplastic lung tissues 8). The expression levels Our studies demonstrated that ADAM28 promotes directly correlated with the MIB-1 (cell prolifera- carcinoma cell proliferation through reactivation of tion marker) labeling index and lymph node insulin-like growth factor-I (IGF-I) by selective metastasis. By in situ hybridization, the signal for digestion of IGF-binding protein-3 (IGFBP-3) of the ADAM28 was observedpredominantlyin the IGF-I/IGFBP-3 complex 11) 12). Another substrate of carcinoma cells andimmunohistochemistry demon- ADAM28 was connective tissue growth factor stratedthat ADAM28 is localizedpredominantlyto (CTGF), andits digestionby ADAM28 ledto release cytoplasm of the carcinoma cells, but a few samples of vascular endothelial growth factor165 (VEGF165) show membrane staining as well as cytoplasmic from the CTGF/VEGF165 complex, causing activation staining. Immunoblotting showedan immunoreac- of angiogenesis 13). ADAM28 also degraded von tive bandof 42 kDa in the carcinoma tissues, but not Willebrandfactor (VWF), which inducedapoptosis in the control lung tissues 8). in human carcinoma cell lines with negligible We developed the ELISA system to measure ADAM28 expression, andthis digestionby

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ADAM28-expressing carcinoma cells enabledthem lung carcinomas. Human antibody inhibitors against to escape from VWF-induced apoptosis 14). Down- ADAM28 wouldbe promising as a target therapy regulation of ADAM28 by siRNA, shRNA or mouse for the patients with non-small cell lung carcinoma. anti-ADAM28 antibody suppressed growth and lung metastasis of intravenously injectedlung References adenocarcinoma cells in nonobese diabetic/severe combinedimmunodeficiency(NOD/SCID) mice. 1) EgebladM, Werb Z: New functions for the matrix This was associatedwith increasedapoptosis of the in cancer progression. Nat Rev Cancer, 2002; 2: 161-174. carcinoma cells within the lung bloodvessels and 2) Shiomi T, Okada Y: MT1-MMP and MMP-7 in invasion suppression of VWF degradation in the blood. From andmetastasis of human cancers. Cancer Metastasis these data, we considered that ADAM28 may be a Rev, 2003; 22: 145-152. 3) Kessenbrock K, Plaks V, Werb Z: Matrix metalloprotei- goodmolecular target for treatment of the patients nases: regulators of the tumor microenvironment. Cell, with non-small cell lung carcinoma 14) 15). 2010; 141: 52-67. 4) Huovila AP, Turner AJ, Pelto-Huikko M, Kärkkäinen I, Ortiz RM: Shedding light on ADAM metalloproteinases. Development of inhibitors against ADAM28 Trends Biochem Sci, 2005; 30: 413-422. and their effects on carcinoma cell 5) Mochizuki S, Okada Y: ADAMs in cancer cell prolifera- proliferationand metastasis tion andprogression. Cancer Sci, 2007; 98: 621-628. 6) Edwards DR, Handsley MM, Pennington CJ: The ADAM metalloproteinases. Mol Aspects Med, 2008; 29: 258-289. To obtain inhibitors against ADAM28 for clinical 7) Blobel CP: ADAMs: key components in EGFR signal- application, we developed human antibodies against ling anddevelopment.Nat Rev Mol Cell Biol, 2005; 6: 32-43. ADAM28 by screening the Human Combinatorial 8) Ohtsuka T, Shiomi T, Shimoda M, et al: ADAM28 is Antibody Library, and obtained several candidate overexpressedin human non-small cell lung carcinomas antibodies. Among them, one antibody was specifi- andcorrelates with cell proliferation andlymph node metastasis. Int J Cancer, 2006; 118: 263-273. cally immunoreactive with ADAM28, andinhibited 9) Kuroda H, Mochizuki S, Shimoda M, et al: ADAM28 is a proteinase activity of ADAM28. The antibody was serological andhistochemical marker for non-small-cell effective to inhibit IGF-I-induced cell proliferation lung cancers. Int J Cancer, 2010; 127: 1844-1856. 10) Abe H, Mochizuki S, Ohara K, et al: Src plays a key role in vitro andlung metastasis in vivo. in ADAM28 expression in v-src-transformedepithelial ADAM28 was originally reportedas lymphocyte- cells andhuman carcinoma cells. Am J Pathol, 2013; 183: specific ADAM 16), but we have recently disclosed 1667-1678. 11) Mochizuki S, Shimoda M, Shiomi T, Fujii Y, Okada Y: that under physiological conditions, ADAM28 is ADAM28 is activatedby MMP-7 (matrilysin-1) and expressed, albeit at low levels, by epithelial cells in cleaves insulin-like growth factor binding protein-3. several human normal tissues including the epididy- Biochem Biophys Res Commun, 2004; 315: 79-84. 12) Mitsui Y, Mochizuki S, Kodama T, et al: ADAM28 is mis, bronchus andstomach, but not by lymphocytes. overexpressedin human breast carcinomas: implica- We have also demonstrated that ADAM28 binds to tions for carcinoma cell proliferation through cleavage of C1q andsuppresses C1q-inducedcelldeathin normal insulin-like growth factor binding protein-3. Cancer Res, 2006; 66: 9913-9920. bronchial epithelial cells, suggesting the first line of 13) Mochizuki S, Tanaka R, Shimoda M, et al: Connective protection against C1q-induced cell damage 17). Since tissue growth factor is a substrate of ADAM28. Biochem both epithelial cells andbloodvessels are sur- Biophys Res Commun, 2010; 402: 651-657. 14) Mochizuki S, Soejima K, Shimoda M, et al: Effect of roundedbysolidbasement membrane, the epithe- ADAM28 on carcinoma cell metastasis by cleavage of von lial cells expressing ADAM28 at a low-level may Willebrandfactor. J Natl Cancer Inst, 2012; 104: 906-922. not be attackedby neutralizing anti-ADAM28 15) Mochizuki S, Okada Y: ADAM28 as a target for human cancers. Curr Pharm Des, 2009; 15: 2349-2358. antibody because the basement membrane blocks 16) Roberts CM, Tani PH, Bridges LC, Laszik Z, Bowditch access of the antibody to the cells. Thus, these data RD: MDC-L, a novel metalloprotease disintegrin cys- suggest that neutralizing antibodies against teine-rich member expressedby human lymphocytes. J Biol Chem, 1999; 274: 29251-29259. ADAM28 may be useful for treatment of the 17) Miyamae Y, Mochizuki S, Shimoda M, et al: ADAM28 is non-small cell lung carcinoma patients in the future. expressedby epithelial cells in human normal tissues In summary, we have demonstrated that andprotects from C1q-inducedcelldeath.FEBS J, 2016; 283: 1574-1594. ADAM28 plays an important role in cancer cell proliferation andmetastasis in human non-small cell

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