1. Defining Met-driven Oncogenic Signaling Pathways in Urologic Malignancies. Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (ccRCC), 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 ccRCC 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 showed that beta-catenin, a junctional protein and gene transactivator, was a critical intracellular mediator of these activities. HGF signaling also contributes to disease progression, tumor invasiveness and metastasis in kidney cancers other than the clear cell type; in particular, MET kinase domain mutations in a hereditary form of papillary renal carcinoma (PRC) are primary drivers of that disease, and Met overabundance is common in both hereditary and sporadic forms. VHL loss of function is rare in PRC, but tumor hypoxia is not; hypoxia strongly enhances HGF-mediated invasiveness and metastasis in a variety of model systems, through largely undefined molecular mechanisms. HGF-driven beta-catenin transcriptional activity is suppressed when VHL is functional, suggesting that the integration of hypoxia and HGF driven cell invasiveness in PRC involves other primary intracellular signaling routes downstream of Met. Over the course of our continued investigation we found that the integration of these signaling pathways involves three parallel routes: (1) hypoxia-induced reactive oxygen species (ROS) production and decreased expression of the mitogen-activated protein kinase (MAPK) negative regulator DUSP2, leading to enhanced cascade activation; (2) ROS-induced diacylglycerol production by phospholipase C leading to protein kinase C activation and increased protein phosphatase-2A activity, thereby suppressing HGF-induced Akt activation; and (3) a profound shift from HGF-enhanced, proliferation-oriented metabolism to autophagy-dependent invasion and suppression of proliferation. These results define the molecular basis of growth factor and hypoxia invasive synergy in VHL-competent papillary RCC cells, illustrate the plasticity of invasive and proliferative tumor cell states and provide signaling profiles by which they may be predicted. 2. Evaluation of pathway antagonists, through structural analysis of HGF/Met interaction and analysis of commercially-developed Met-targeting agents. In collaboration with Amgen scientists that developed HGF- and Met-targeted agents, we developed a model of acquired drug resistance to rilotumumab, a fully human HGF-neutralizing monoclonal antibody now in phase II and III clinical trials. Acquired drug resistance is a long-standing problem of cancer therapeutics, and this issue has become even more vexing with the development of highly selective targeted agents. Anticipating acquired resistance and understanding its basis should help develop clinical strategies to prevent or circumvent its occurrence. We developed our model of rilotumumab resistance using glioblastoma multiformae (GBM)-derived cells, because HGF/Met signaling is a likely contributor to oncogenesis and tumor progression in that disease. In anticipation of acquired resistance to rilotumumab, models of acquired resistance were generated by growing GBM-derived U87 MG cells in maximally effective drug concentrations and by low dose treatment of mice implanted with U87 MG cells. Remarkably, in both approaches rilotumumab resistant cell lines and tumors remained sensitive to a selective Met tyrosine kinase inhibitor, and therefore dependent on HGF/Met signaling. Resistant cell lines developed in vitro and cell lines derived from rilotumumab resistant mouse xenografts uniformly displayed dramatic HGF overproduction which triggered chronic endoplasmic reticulum (ER) stress and the unfolded protein response. These events in turn slowed the progression of HGF and Met from the ER and Golgi apparatus, enhancing their interaction and Met activation in subcellular compartments that are inaccessible to rilotumumab. The absence of HGF or MET mutation and retention of HGF pathway dependence observed in our model is unlike many other mechanisms of acquired drug resistance, where mutation of the gene encoding the drug target (e.g. EGFR), loss of prominent negative regulators downstream of the drug target, or activation of alternative mitogenic pathways parallel to the target are prevalent means of restoring tumor cell proliferative and invasive activities. The model is similar to others in that restoration of signaling via the PI3K and MAPK pathways is ultimately achieved, whether resistance was acquired to neutralizing antibodies or small molecule kinase inhibitors. Despite the apparent complexity of the mechanism of acquired resistance to rilotumumab, its relatively rapid development suggests that GBM cells in which HGF is an important oncogenic driver are predisposed to use it. Measuring HGF/Met pathway activity in GBM patients is a logical basis for selecting those most likely to benefit from HGF/Met-targeted therapeutics; our results further suggest that monitoring Met pathway activity and HGF production in those patients could provide early indications of acquired resistance, that Met kinase inhibitors may still be efficacious when resistance occurs, and that targeting critical mediators of resistance may provide effective alternative or combinatorial treatments. 3. Development of HGF/Met-related reagents and methods for diagnosis, pharmacodynamics, patient selection and molecular imaging. 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 two site 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. This work has recently extended to bladder cancer, where it was found that urinary shed Met ectodomain levels varied directly with tumor grade and stage, suggesting that testing for urinary shed Met could be useful to identify those bladder cancer patients with a greater likelihood of responding to inhibitors of the HGF/Met pathway. In collaboration with the CCR Molecular Imaging Program (Dr. Peter Choyke) we obtained the recombinantly engineered one-armed monoclonal anti-Met antibody OA-5D5 (MetMab), as well as Met TK inhibitors as ancillary reagents, from Genentech to develop MetMab for PET imaging. Initial studies confirm the feasibility of using MetMab for micro-PET imaging of Met-expressing human tumor xenografts in mice. Future studies should provide improved signal-to-noise ratio and increased sensitivity of Met detection. In parallel, we have obtained Met ectodomain-selective peptides from GE Healthcare for development as imaging agents. Preliminary studies using various cancer cell lines implanted as xenografts show increased tumor labeling in direct proportion to Met expression level.
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