Treatment with Halofuginone Results in Marked Growth Inhibition of a von Hippel-Lindau Pheochromocytoma in Vivo David J. Gross, 1 Israel Reibstein, Lola Weiss, Shimon Slavin, Hagit Dafni, Michal Neeman, Mark Pines, and Arnon Nagler Endocrinology and Metabolism Service [D. J. G.] and Department of Bone Marrow Transplantation and Cancer Immunobiology [I. R., L. W., S. S.], Hadassah University Hospital, Jerusalem 91120; Department of Biological Regulation, Weitzmann Institute of Science, Rehovot 76100 [H. D., M. N.]; Institute of Animal Science, The Volcani Center, Bet Dagan [M. P.]; and Department of Bone Marrow Transplantation, Sheba Medical Center, Tel-Hashomer [A. N.], Israel ABSTRACT Halofuginone has recently been shown to inhibit tumor progression of various types of cancers. The antitumoral effect was associated with decreased tumor angiogenesis rather than a direct cytostatic effect on the tumor cells. The antiangiogenic action of the drug could be related to its inhibition of collagen type I synthesis, inhibition of matrix metalloproteinases (MMPs), or via both mechanisms be- cause both collagen synthesis and MMP activity have been shown to be involved in angiogenesis. Vascular endothelial growth factor (VEGF), in addition to its effect on endothelial cell proliferation, has been shown to be a potent inducer of MMP expression. Because von Hippel-Lindau (VHL)-asso- ciated tumors express high levels of VEGF, it was of interest to ascertain the potential usefulness of halofuginone for treatment of these tumors. Pheochromocytoma tissue frag- ments obtained at surgery from a VHL type 2a patient were propagated s.c. in male BALB/c / (nude) mice. For exper- iments, 2–3-mm tumor fragments were transplanted second- arily s.c. to nude mice. Two treatment groups received halofuginone in standard lab chow at 3 and 5 ppm; control animals received regular chow. All groups were followed for 6 weeks after transplantation. A marked and significant diminution of tumor size and weight was observed in the drug-treated animals (>90% reduction of mean tumor vol- ume for both the 3 and 5 ppm groups). In vivo magnetic resonance imaging analysis of tumors in halofuginone- treated animals showed a significant reduction of vascular functionality. Immunohistochemical studies revealed de- creased collagen type I levels and vascular density in treated tumors and gelatinase assays of tumor extracts revealed a reduction of MMP-2 and MMP-9 activity in halofuginone- treated cells. Taken together, our data indicate that therapy directed at blocking MMP activity (presumably related to excessive VEGF expression in VHL) and reduction of type I collagen deposition curtails angiogenesis and thereby pre- sumably tumor growth in this model system. INTRODUCTION VHL 2 disease is an autosomal dominant cancer syndrome characterized clinically by retinal angiomas, cerebellar and spi- nal cord hemangioblastomas, RCCs, pheochromocytomas, and less commonly, pancreatic islet cell tumors and epididymal cysts. This multiorgan involvement can appear in various com- binations; however, in an affected kindred, the clinical manifes- tations of the disease are usually invariable and breed true. The gene associated with VHL has been cloned, and germ-line mutations in the VHL gene can be detected in virtually all patients with VHL: these can be micro- and macrodeletions or point mutations scattered throughout all three exons of the VHL gene. The VHL gene encodes a 213 amino acid peptide, pVHL. pVHL, considered to be a tumor suppressor gene, has pleotropic effects in the cell, the most prominent being the lack of cellular responsiveness to ambient oxygen tension. Thus, it has been shown that in tumor cell lines derived from VHL patients, mutated pVHL is associated with constitutively elevated VEGF mRNA, unresponsive to normal normoxia/hypoxia regulation. Reintroduction of wild-type pVHL results in restoration of VEGF regulation and inhibition of tumor growth in vivo (for review see Ref. 1). Thus, mutated pVHL-related overexpression of VEGF and its receptors in the well-vascularized VHL tumors might implicate unregulated VEGF-induced angiogenesis as a major pathogenetic pathway, leading toward tumor formation in this disease. We have recently shown that the antiangiogenic agent linomide significantly inhibits the growth of VHL pheo- chromocytoma explants in nude mice (2). This effect was found to be associated with decreased tumor vascularity and VEGF mRNA levels. Our data indicated that in VHL disease, therapy directed at abrogation of the effect of constitutively expressed VEGF may constitute an effective medical treatment. Because the clinical use of linomide is precluded by unacceptable tox- icity in human subjects (3), we sought an alternative antiangio- genic approach. Halofuginone has recently been shown to in- hibit tumor progression of C6 glioma, bladder carcinoma, and prostate tumors (4 – 6). In these tumors, a correlation between the inhibition of tumor growth and reduction in collagen type I synthesis was observed. This antitumoral effect has been shown Received 1/7/03; revised 4/7/03; accepted 4/9/03. 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 To whom requests for reprints should be addressed, at Present address: Institute of Endocrinology and Metabolism, Rabin Medical Center, (Beilinson Campus), Petach-Tikva 49100, Israel. Phone: 972-3- 9377184; Fax: 972-3-9211403; E-mail: gross@vms.huji.ac.il. 2 The abbreviations used are: VHL, von Hippel-Lindau; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; MRI, magnetic resonance imaging; ECM, extracellular matrix; EC, endothe- lial cell; RCC, renal cell carcinoma. 3788 Vol. 9, 3788 –3793, September 1, 2003 Clinical Cancer Research Research. on April 25, 2018. © 2003 American Association for Cancer clincancerres.aacrjournals.org Downloaded from