Z01 BC 011124 (Z01) | |||
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Title | Oncogenic Met Signaling in Urologic Malignancies | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Bottaro, Donald | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $181,253 | Project Dates | 10/01/2007 - N/A |
Fiscal Year | 2008 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Breast Cancer (20.0%) Cervical Cancer (10.0%) Esophagus (10.0%) Kidney Cancer (100.0%) Kidney Disease (100.0%) Lung (10.0%) Melanoma (10.0%) Ovarian Cancer (10.0%) Prostate (50.0%) Skin (10.0%) Wilm's Tumor (10.0%) |
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Research Type | |||
Cancer Initiation: Oncogenes and Tumor Suppressor Genes Systemic Therapies - Discovery and Development |
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Abstract | |||
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. VHL loss has been shown to allow 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. We 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, a collaborative effort to investigate other activators of beta-catenin signaling in RCC, such as Wnt family proteins, and a systematic survey of beta-catenin mediation of HGF-driven invasiveness in other urologic malignancies. 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. Results described in a manuscript in preparation demonstrate that specific HS binding interactions are critical for HGF-stimulated cell motility, proliferation and tumor growth, 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 by Virtual Screening 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. To identify novel Met TK inhibitors with minimal experimental testing, we used a virtual screen to identify new lead compounds that specifically inhibit the Met TK in its inactive state. The goal of virtual high-throughput screening is to test compounds computationally instead of physically. Our screening method included filtering of a large database of commercially available compounds (> 13 million) based on physicochemical properties, receptorligand docking and scoring, and pharmacophore searches within the docking results. This produced an initial subset of approximately 600,000 compounds, which was reduced to a final set of 175 with very little structural similarity to any known kinase inhibitors. The top 70 compounds were tested in a cell-free assay system and in intact cells. In a manuscript now in preparation, we describe three compounds that showed inhibition of Met at micromolar or submicromolar levels. 4. Preclinical assessment of third party Met-targeting agents 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. Ongoing collaborations to aid in the preclinical assessment of other third party Met-targeting agents for kidney, prostate and bladder cancer have been established with Genentech, GlaxoSmithKline, GE Healthcare, Johnson and Johnson, Novartis, and Pfizer. 5. 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, include the further development of the Met TK assay as a diagnostic tool for patient selection and monitoring drug efficacy. |