[CANCER RESEARCH 61, 1022–1028, February 1, 2001] Metastatin: A Hyaluronan-binding Complex from Cartilage That Inhibits Tumor Growth 1 Ningfei Liu, 2 Randall K. Lapcevich, 2 Charles B. Underhill, Zeqiu Han, Feng Gao, Glenn Swartz, Stacy M. Plum, Lurong Zhang, 2 and Shawn J. Green 2, 3 Department of Oncology, Georgetown University Medical Center, Washington, D.C. 20007 [N. L., C. B. U., Z. H., F. G., L. Z.], and EntreMed, Inc., Rockville, Maryland 20902 [R. K. L., G. S., S. M. P., S. J. G.] ABSTRACT In this study, a hyaluronan-binding complex, which we termed Met- astatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo bio- logical effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability to bind hyaluronan on the target cells. Collectively, these results suggest that Metastatin is an effective antitumor agent that exhibits antiangiogenic activity. INTRODUCTION A potential therapeutic target on angiogenic endothelial cells is hyaluronan, a large negatively charged glycosaminoglycan that plays a role in the formation of new blood vessels (1). Particularly high concentrations of hyaluronan are associated with endothelial cells at the growing tips or sprouts of newly forming capillaries (2, 3). Similarly, when cultured endothelial cells are stimulated to proliferate by cytokines, their synthesis of hyaluronan is significantly increased (4). Interestingly, this stimulation is restricted to endothelial cells derived from the small blood vessels and is not seen in endothelial cells derived from larger ones (4). In the case of mature blood vessels, hyaluronan is present in perivascular regions and in the junctions between the endothelial cells (5, 6). Earlier studies have shown that exogenously applied hyaluronan has different effects on angiogenesis depending on its size, with macromolecular hyaluronan inhibiting vascularization in chicken embryos, and oligosaccharide fragments of hyaluronan stimulating vascularization in the chorioallantoic mem- brane (7–9). Thus, hyaluronan appears to be specifically associated with the endothelial cells of newly forming blood vessels and can influence their behavior. In addition to hyaluronan, endothelial cells involved in neovascu- larization also express CD44 and other cell surface receptors for hyaluronan (10 –12). In particular, endothelial cells associated with tumors express large amounts of CD44 (11). In previous studies, we have shown that CD44 allows cells to bind hyaluronan so that it can be internalized into endosomal compartments, where the hyaluronan is degraded by the action of acid hydrolases (13, 14). Thus, the expression of CD44 by endothelial cells allows them to bind and internalize hyaluronan as well as any associated proteins. The fact that both hyaluronan and CD44 are up-regulated in endothelial cells in- volved in neovascularization suggests that the turnover of hyaluronan by these cells is much greater than that by cells lining mature blood vessels. The increased turnover of hyaluronan in tumor-associated endothe- lial cells suggested a possible mechanism to specifically target these cells. Our initial idea was to use a hyaluronan-binding complex isolated from cartilage to deliver chemotherapeutic agents specifically to these endothelial cells. Purified by affinity chromatography, this hyaluronan-binding complex consists of tryptic fragments of the link protein and aggrecan core protein (5, 15, 16). We intended to couple the hyaluronan-binding complex to a chemotherapeutic agent such as methotrexate and use this derivative to attack endothelial cells. We hoped that this derivative would bind to the hyaluronan on the endothelial cells and then be internalized into lysosomes, where the methotrexate would be released by the action of acid hydrolyses. Surprisingly, however, in the course of these experiments, we found that the hyaluronan-binding complex by itself (i.e., in the absence of a chemotherapeutic agent) inhibited angiogenic activity. Functionally, we termed the hyaluronan-binding complex, which inhibits tumor growth, Metastatin. In the present study, we demonstrate that Metastatin has a number of intriguing biological activities, including inhibition of endothelial cell proliferation and migration, inhibition of angiogenesis, and sup- pression of tumor cell growth in chicken embryos and pulmonary metastasis in mice. These effects are blocked by preincubating Met- astatin with hyaluronan, suggesting that the activity of Metastatin depends on its ability to bind hyaluronan on the target cells. MATERIALS AND METHODS Preparation of Metastatin. The hyaluronan-binding complex was pre- pared by a modified version of the method originally described by Tengblad (15, 16). Briefly, bovine nasal cartilage (Pel-Freez, Rogers, AR) was shredded with a Sure-Form blade (Stanley), extracted overnight with 4 M guanidine-HCl and 0.5 M sodium acetate (pH 5.8), and dialyzed against distilled water to which 10 PBS was added to a final concentration of 1 PBS (pH 7.4). The protein concentration was measured, and for each 375 mg of protein, 1 mg of trypsin (type III; Sigma, St. Louis, MO) was added. After digestion for 2 h at 37°C, the reaction was terminated by the addition of 2 mg of soybean trypsin inhibitor (Sigma) for each milligram of trypsin. The digest was dialyzed against 4 M guanidine-HCl and 0.5 M acetate (pH 5.8), mixed with hyaluronan coupled to Sepharose, and then dialyzed against a 10-fold volume of distilled water. The hyaluronan-Sepharose beads were placed into a chromatography column and washed with 1.0 M NaCl, followed by a gradient of 1.0 –3.0 M NaCl. Metastatin was eluted from the hyaluronan affinity column with 4 M guanidine-HCl and 0.5 M sodium acetate (pH 5.8), dialyzed against saline, and sterilized by passage through a 0.2-m-pore filter. For SDS-PAGE analysis, the purified preparation was loaded onto a 10% BisTris nonreducing gel (Novex, Inc.) and subsequently stained with Coomassie Blue. To identify the Received 7/12/00; accepted 11/28/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by the United States Army Medical Research and Materiel Command under DAMD1717-94-J-4284, DAMD17-98-1-8099, and DAMD17-99-1- 9031. Additional support was obtained from the Susan G. Komen Foundation and NIH Grant R29CA71545. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at EntreMed, Inc., Medical Center Drive, Suite 200, Rockville, MD 20902. Phone: (301) 738-2494; Fax (301) 217- 9594; E-mail shawng@entremed.com. 1022 Research. on January 22, 2016. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from