ORIGINAL PAPERS Cleavage of metastasis suppressor gene product KiSS-1 protein/metastin by matrix metalloproteinases Takahisa Takino 1 , Naohiko Koshikawa 2 , Hisashi Miyamori 1 , Motohiro Tanaka 3 , Takuma Sasaki 3,4 , Yasunori Okada 5 , Motoharu Seiki 2 and Hiroshi Sato* ,1,4 1 Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan; 2 Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; 3 Department of Chemotherapy, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan; 4 Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan; 5 Department of Pathology, Medical School, Keio University, Tokyo 160-8582, Japan A human placenta cDNA library was screened by the expression cloning method for gene products that interact with matrix metalloproteinases (MMPs), and we isolated a cDNA whose product formed a stable complex with pro- MMP-2 and pro-MMP-9. The cDNA encoded the metastasis suppressor gene KiSS-1. KiSS-1 protein was shown to form a complex with pro-MMP. KiSS-1 protein is known to be processed to peptide ligand of a G-protein- coupled receptor (hOT7T175) named metastin, and sup- presses metastasis of tumors expressing the receptor. Active MMP-2, MMP-9, MT1-MMP, MT3-MMP and MT5- MMP cleaved the Gly 118 -Leu 119 peptide bond of not only full-length KiSS-1 protein but also metastin decapeptide. Metastin decapeptide induced formation of focal adhesion and actin stress fibers in cells expressing the receptor, and digestion of metastin decapeptide by MMP abolished its ligand activity. Migration of HT1080 cells expressing hOT7T175 that harbor a high-level MMP activity was only slightly suppressed by either metastin decapeptide or MMP inhibitor BB-94 alone, but the combination of metastin decapeptide and BB-94 showed a synergistic effect in blocking cell migration. We propose that metastin could be used as an antimetastatic agent in combination with MMP inhibitor, or MMP-resistant forms of metastin could be developed and may also be efficacious. Oncogene (2003) 22, 4617–4626. doi:10.1038/sj.onc.1206542 Keywords: MMP; KiSS-1; cleavage Introduction Matrix metalloproteinases (MMPs) are a family of Zn 2 þ -dependent enzymes that are known to cleave extracellular matrix (ECM) proteins in normal and pathological conditions (Woessner and Gunja, 1991; Birkedal et al., 1993; Stetler-Stevenson et al., 1993). To date, 21 mammalian MMPs have been identified by cDNA cloning and they can be subgrouped into 15 soluble-type and six membrane-type MMPs (MT- MMPs) (Nagase and Woessner, 1999; Seiki, 1999). MMPs are overexpressed in various human malignan- cies and have been thought to contribute to tumor invasion and metastasis by degrading ECM components (Birkedal et al., 1993; Stetler-Stevenson et al., 1993). Thus, the level of MMP expression correlates with the invasiveness or malignancy of tumors (Nomura et al., 1995; Ueno et al., 1997). Particularly, MT1-MMP, MMP-2, MMP-7 and MMP-9 have been reported to be most closely associated with tumor invasion and metastasis. While degradation of ECM is an important aspect of MMP biology, growing evidence demonstrated specific processing/activation or degradation of cell surface receptors and ligands. Fas ligand (Powell et al., 1999), TNF-a (Gearing et al., 1994), the ectodomain of the fibroblast growth factor receptor-1 (Levi et al., 1996), the heparin-binding epidermal growth factor (Suzuki et al., 1997) and IL-8 (Van den Steen et al., 2000) were reported to be released or activated by MMPs. And MMPs cleave and inactivate IL-1b (Ito et al., 1996), insulin-like growth factor binding proteins (Fowlkes et al., 1994), fibrinogen and factor XII (Hiller et al., 2000), the CC chemokine MCP-3 (McQuibban et al., 2000), and the CXC chemokines stromal-cell- derived factor (SDF)-1a and -b (McQuibban et al., 2001, 2002). The KiSS-1 gene was identified originally as being differentially upregulated in C8161 melanoma cells that have lost the potential to metastasize after microcell- mediated transfer of human chromosome 6 (Lee et al., 1996; Lee and Welch, 1997). The exogenous expression of KiSS-1 in breast carcinoma cells also prevents these cells from metastasizing (Lee et al., 1997). Stable transfection of KiSS-1 into HT1080 cells was reported to downregulate the expression of MMP-9 (Yan et al., 2001). It was also demonstrated that loss of KiSS-1 mRNA expression correlates with progression of human Received 20 November 2002; revised 24 February 2003; accepted 24 February 2003 *Correspondence: H Sato, Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Japan; E-mail: vhsato@kenroku.kanazawa-u.ac.jp Oncogene (2003) 22, 4617–4626 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc