A hepatocyte growth factor antagonist engineered by site-directed mutagenesis.

Abstract

10528 Background: 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. Methods: To understand the role of HS in ligand-receptor interaction and biological signaling, we identified three putative HS binding residues in N domain (K60, K62 and R73), generated opposite charge mutations at these positions in NK1, and characterized the recombinantly expressed proteins biochemically and biologically. Results: Nuclear magnetic resonance analysis of mutant and wild type (WT) proteins revealed that no significant structural alterations were introduced by the mutations. The binding of each of the mutant proteins to immobilized heparin was reduced relative to WT, most profoundly in the combined triple mutant (3M). 3M also lacked biological activity, and competitively antagonized HGF activity in normal cells. 3M also potently inhibited oncogenic Met signaling. Soft agar colony formation by HGF-driven tumor cell lines was reduced 50% in the presence of 70 nM 3M protein and abolished in the presence of 300 nM, relative to untreated controls. Similar levels of inhibition were observed using the Met inhibitor PHA665752 at the same concentrations. Ectopic 3M expression in these tumor lines dramatically reduced their growth rate as xenografts in mice and was associated with reduced tumor phosphoMet content. HGF-driven experimental metastasis by B16 cells was blocked by ectopic 3M expression, or by daily intraperitoneal (IP) administration of purified recombinant 3M. Conclusions: Our results show that HS binding is critical for HGF-driven mitogenesis, motogenesis, tumor growth and metastasis, and inhibition of these activities by 3M validates a novel strategy for developing antagonists of the HGF signaling pathway and potentially those of other HS-binding ligands.