Abstract 342: A hepatocyte growth factor antagonist engineered by disruption of heparan sulfate binding

Abstract

Abstract Hepatocyte growth factor (HGF) stimulates cell proliferation, motility, and morphogenesis upon binding to the receptor tyrosine kinase Met and cell surface heparan sulfate (HS) glycans. NK1 is a truncated HGF isoform consisting of the N-terminal (N) and first kringle (K1) domains of full-length HGF that stimulates all major HGF biological activities. Within NK1, the N domain contains the HS binding site, while K1 contains the primary determinants of Met binding. To further define the role of HS in ligand-receptor interaction and biological signaling, we identified the primary HS binding residues in N domain (K60, K62, R73) and generated opposite charge mutations at these positions in NK1. Nuclear magnetic resonance analysis of recombinantly expressed mutant and wild type (WT) proteins revealed that both were folded normally. Mutant proteins displayed diminished binding to immobilized HS relative to WT, and the triple mutant protein (3M) also lacked intrinsic motogenic and mitogenic activity. Interestingly, 3M NK1 maintained Met binding and competitively inhibited DNA synthesis stimulated by WT NK1 as well as full-length HGF in normal cells. Expression plasmids encoding 3M and WT NK1 proteins were transfected into the glioblastoma cell U87 MG, which is dependent on HGF/Met autocrine signaling for tumorigenicity in mice. 3M/U87 MG cells displayed a substantially lower rate of growth relative to WT/U87 MG or the parental cell line, suggestive of growth antagonism by 3M NK1. As anticipated, expression of WT NK1 significantly accelerated the growth of U87 MG tumor xenografts in mice, which reached maximal volume (1 ml) by 22 days post-implantation, compared to 45 days required by the parental cell line. In contrast, tumor growth in animals injected with 3M/U87 MG was dramatically impaired: at 60 days post implantation, average tumor volume for this group was < 0.3 ml. Because both WT and 3M transfectants expressed equal amounts of their respective recombinant proteins, the HS binding site mutations delayed measurable tumor growth from one week to two months. Thus HS binding is critical for HGF-driven mitogenesis and tumor growth. We now show that HGF signaling requires specific HS binding interactions and that targeted disruption of these HS binding sites in HGF yields a potent and selective competitive inhibitor of normal and oncogenic HGF signaling. Our findings further define the role of HS in growth factor signaling and reveal a novel strategy for antagonist development which may be applicable to other HS-binding growth factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 342.