ADAMTS4 and its proteolytic fragments differentially affect melanoma growth and angiogenesis in mice Nithya Rao 1 , Zhiyuan Ke 1 , Hongrui Liu 2 , Chao-Jin Ho 1 , Saran Kumar 1 , Wei Xiang 1 , Yizhun Zhu 2 and Ruowen Ge 1 1 Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore 2 Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China The metalloproteinase ADAMTS4 (ADAMTS, a disintegrin-like and metalloproteinase with thrombospondin motif)/aggrecanase- 1 is highly expressed in cartilage and has been implicated in human arthritis. Although abundantly expressed in many types of cancer, its role in cancer remains unknown. In this work, we demonstrate for the first time that full-length ADAMTS4 and its catalytically more active N-terminal 53 kDa autocatalytic fragment both promote B16 melanoma growth and angiogenesis in mice. In contrast, overexpression of its catalytically inactive E362A mutant or truncated fragments containing only the C-termi- nal ancillary domains suppresses melanoma growth and angiogenesis under similar conditions. Structure–function mapping revealed that the single thrombospondin-type 1 repeat domain is essential and sufficient for the antitumorigenic activity dis- played by the catalytically inactive ADAMTS4 isoforms. Suppression of tumor growth and angiogenesis in mice is accompanied by a significant increase in tumor cell apoptosis, whereas tumor cell proliferation is not affected. Importantly, we identified and demonstrated the presence of novel proteolytic fragments of ADAMTS4 containing essentially only the C-terminal ancillary domains in cultured cells, and also in human cancer tissues, coexisting with full-length and catalytically active N-terminal frag- ments. The contrasting functions toward tumor growth in mice by the wild-type proteinase and its catalytically inactive mutant correlate with their contrasting influences on angiogenesis signaling pathway molecules in B16 melanoma in mice. Our results suggest a complex role for ADAMTS4 in cancer with the functional balance of protumorigenic and antitumorigenic isoforms likely to act as an important parameter in determining the net influence of this metalloproteinase on tumor growth in vivo. The ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin motifs) is a family of extracellular metallo- proteinases mediating diverse functions including matrix deg- radation, blood coagulation and angiogenesis. 1,2 The thrombospondin-type 1 repeat (TSR), a common motif that exists among the members of this family of proteinases, plays an important role in regulating angiogenesis. 3 The founding member of this family, ADAMTS1, was first reported to be an antiangiogenic protein along with ADAMTS8 as it sup- pressed basic fibroblast growth factor and vascular endothe- lial growth factor (VEGF)-induced angiogenesis. 4 ADAMTS4 is closely related to ADAMTS1 phylogenetically with 64% similarity based on full-length protein sequence. Recently, several other members of the ADAMTS family such as ADAMTS2, 5 ADAMTS4, 6 ADAMTS5, 7,8 ADAMTS9 9 and ADAMTS12, 10 have also been demonstrated to have an angiostatic function either in vitro or both in vitro and in vivo, in addition to the previously identified ADAMTS1 and ADAMTS8. 4 ADAMTS4 is the only member of this family with a single centrally located TSR motif and no TSRs at the C-terminus. It has been well characterized for its role as an aggrecanase, involved in cartilage destruction in human rheumatoid ar- thritis and osteoarthritis. 11 However, ADAMTS4-null mice showed no protection against aggrecan degradation in inflammatory or surgically induced arthritis in mice, 12,13 indi- cating that ADAMTS4 is not the main aggrecanase in mouse arthritis. The 837 amino acid long human ADAMTS4 protein con- tains a long signal peptide, a prodomain, a metalloproteinase catalytic domain with zinc-binding motif, a disintegrin-like domain, a central TSR motif, followed by a cysteine-rich region with ten conserved cysteine residues, and a spacer do- main (Fig. 1). ADAMTS4 is synthesized as an inactive zymo- gen. Activation of the protein by furin-mediated cleavage occurs in trans-Golgi network 14 and the mature protein is secreted into the extracellular matrix (ECM). In ECM, ADAMTS4 undergoes further C-terminal cleavage at Lys 694 - Phe 695 and Thr 581 -Phe 582 , respectively, to generate two other Key words: ADAMTS4, tumorigenesis, angiogenesis, metalloprotei- nase, TSR Additional Supporting Information may be found in the online version of this article. Grant sponsor: The Singapore Biomedical research Council; Grant number: BMRC/01/1/21/18/074; Grant sponsor: Ministry of Education; Grant number: R-154-000-447-112 DOI: 10.1002/ijc.28037 History: Received 11 May 2012; Accepted 20 Dec 2012; Online 15 Jan 2013 Correspondence to: Ruowen Ge, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore, Tel.: 165-65167879, Fax: 165-67792486, E-mail: dbsgerw@nus.edu.sg Cancer Cell Biology Int. J. Cancer: 133, 294–307 (2013) V C 2013 UICC International Journal of Cancer IJC