1. Characterization of Met-driven Oncogenic Signaling in Urologic Malignancies. Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. We previously found that VHL loss allows robust RCC cell motility, invasiveness and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential, and identified beta-catenin, a junctional protein and gene transactivator, as a critical intracellular mediator of these activities. Reconstitution of VHL expression in RCC cells repressed HGF-stimulated beta-catenin tyrosyl phosphorylation, adherens junction disruption, cytoplasmic beta-catenin accumulation and reporter gene transactivation. Ectopic expression of a ubiquitination resistant beta-catenin mutant specifically restored HGF-stimulated invasion and morphogenesis in VHL transfected RCC cells. Finally, VHL gene silencing in non-RCC renal epithelial cells phenotypically mimicked VHL loss in RCC, and HGF driven invasiveness was blocked by the expression of a dominant negative mutant of the beta-catenin transcriptional co-activator, Tcf. These findings identify beta-catenin as a potential target in biomarker and drug development for RCC. Ongoing work in this area includes histopathological analysis of human RCC specimens to assess beta-catenin localization, and a collaborative effort to investigate other activators of beta-catenin signaling in RCC, such as Wnt family proteins. 2. Structure-Based Design of HGF/Met Pathway Antagonists Structurally, HGF resembles plasminogen;the two proteins share almost 40% amino acid sequence identity and several structural motifs. HGF is also a heparin-binding growth factor, a property that influences its biodistribution, stability, and biological activity. HGF pathway activation begins with HGF binding to the Met receptor tyrosine kinase and to cell surface heparan sulfate (HS) proteoglycans. The importance of HS binding for various HGF biological responses remains controversial. The HGF gene encodes full-length HGF, and two truncated isoforms (NK1 and NK2) which consist of the N-terminal domain (N) linked to the first one (K1) or two (K1+K2) kringle domains. Both truncated isoforms are motogenic;NK1 also retains mitogenic and morphogenic activities. Within NK1, the N domain has the HS binding activity of full-length HGF and can self-associate in solution, a property which is enhanced in the presence of added HS or heparin. K1, in contrast, does not bind tightly to HS or heparin, but has critical determinants for Met interaction. To better define the role of HS in HGF biological responses, HS binding residues in the N domain were replaced by residues of opposite charge in the context of NK1. Recombinant mutant and wild type NK1 proteins were purified and analyzed structurally by nuclear magnetic resonance (NMR) spectroscopy, biochemically for HS and Met binding, and biologically using cultured cell-based assays and tumorigenicity studies in mice. Our results demonstrate that specific HS binding interactions are critical for HGF-stimulated cell motility, proliferation and tumor growth, shed light on the mechanism of ligand-induced receptor activation, and reveal a novel strategy for the design of potent and selective antagonists of HS-dependent oncogenic growth factor signaling pathways. 3. Identification of Novel Met TK Inhibitors and Evaluation of Third-party Met Pathway Antagonists The overall structure of the Met receptor is that of a typical RTK, with an extracellular ligand binding domain, a transmembrane helix, and an intracellular kinase domain. The latter has the standard kinase fold, with an amino-terminal beta-sheet-containing lobe and a carboxyl-terminal helical lobe connected through a hinge region. The ATP binding site is in a deep, narrow, coin-slot-like cleft between the two lobes. In prior work a virtual screen was used to identify novel Met TK inhibitors with minimal experimental testing. Our method included filtering of a large database of commercially available compounds (>13 million) based on physicochemical properties, receptor-ligand docking and scoring, and pharmacophore searches within the docking results. This produced a final set of 175 compounds with very little structural similarity to any known kinase inhibitors. The top 100 compounds were tested in cell-free and intact-cell based assay systems, leading to the identification of three compounds that inhibited Met kinase activity at micromolar or submicromolar levels. These compounds are now in a second round of design optimization by combined in silico and conventional structure-activity relationship studies. In collaboration with Dr. Teresa Burgess and colleagues at Amgen, we mapped the binding epitope of the experimental anti-cancer therapeutic AMG102, a neutralizing monoclonal antibody against HGF now in phase 2 clinical trials. The results reveal a novel epitope of HGF/Met interaction that is essential for HGF-driven pathway activation, and reveal a potential route to drug resistance through upregulated expression of truncated HGF isoforms. A drug-resistant, tumor-derived cell line has been developed to further define the mechanism by which resistance occurs, with the goal of identifying effective strategies to counter and/or circumvent resistance to AMG102. Ongoing collaborations to aid in the preclinical assessment of other third party Met-targeting agents to treat and/or diagnose kidney, prostate and bladder cancer have been established with Genentech, GlaxoSmithKline, GE Healthcare, Johnson and Johnson, Novartis, and Pfizer. 4. Diagnostic and prognostic assay development. Many proteins are proteolytically released from the cell surface by a process known as ectodomain shedding. Shedding occurs under normal physiologic conditions and can be increased in certain pathologies. We hypothesized that Met overexpression in cancer might result in increased ectodomain shedding and that its measure could be a useful biomarker of tumor progression. We developed a sensitive electrochemiluminescent immunoassay to quantitate Met protein in cell lysates, culture supernatants and biological samples. A survey of cultured cell lines revealed a direct correlation between malignant potential and Met shedding rate. Plasma and urine samples from mice harboring subcutaneous human tumor xenografts displayed soluble human Met levels that correlated directly with tumor volume, suggesting that Met ectodomain shedding may provide a reliable and practical indicator of malignant potential and overall tumor burden. To further investigate this possibility, we have established a series of collaborations to measure soluble Met in human urine and plasma samples obtained from patients with RCC, prostate and bladder cancer, as well as other malignancies. We have also adapted this assay to measure Met TK activation in cultured cells, which was the basis for our biochemical Met TK inhibitor screen. Additional refinement has allowed us to measure Met expression and activation levels, in absolute units, in human tissue specimens. Many of the collaborations with industrial partners that have developed Met-targeted agents as anti-cancer drugs, as described above, includ [summary truncated at 7800 characters]
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