1. Defining the roles of EGFR and Met pathway components in RCC. Clear cell renal cell carcinoma (RCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant transcriptional activation of genes that contribute to tumor growth and metastasis, including transforming growth factor-alpha (TGF-alpha), a ligand of the epidermal growth factor receptor (EGFR) tyrosine kinase. Autocrine TGF-alpha/EGFR pathway activation in VHL-negative clear cell RCC results in overexpression of the EGFR receptor in RCC compared to normal renal tissue and constitutive activation of certain EGFR pathway effectors, contributing to cell survival, proliferation, and invasiveness. Constitutive Met activation also occurs in clear cell RCC, although its basis is not completely understood. Because the MET gene is a transcriptional target of this pathway, even partial constitutive Met activation is likely to contribute to the Met protein overexpression observed in clear cell RCC. Prior studies from this project have shown that among the known EGFR effectors, Akt-1, a serine/threonine kinase activated downstream of phosphatidylinositol 3-kinase (PI3K), and MEK-1, a member of the extracellular signal-regulated kinase (ERK) serine/threonine kinase family, are critical mediators of cell survival and oncogenicity in RCC cells in culture. These effects are shared by the Met signaling pathway, and aberrant activation of both pathways simultaneously is likely to contribute to an aggressive clear cell RCC phenotype. Interestingly, matrix invasion was found to be a better predictor of tumorigenicity then cell proliferation, highlighting the value of invasion assays as a predictor of tumorigenesis for RCC and revealing that even partial silencing of Akt-1 or MEK-1 can produce striking changes in RCC cell invasiveness amidst persistent survival and growth signaling. This is particularly relevant to targeted anti-cancer therapies because it suggests that complete silencing of certain genes that may cause unacceptable systemic toxicity may not be required for effective disease control. 2. Preclinical assessment of third party third party inhibitors of the VHL/HIF/mTor axis as research tools and to assess their preclinical efficacy. In addition to mRNA knockdown, this project incorporates the study of available pharmacological agents targeting EGFR, Met and other VHL/HIF/mTor-regulated oncogenic pathways. These include gefitinib (Iressa, Astra Zeneca), the experimental drug candidates ZD6474 (Astra Zeneca), targeting both the EGFR and vascular endothelial growth factor receptor (VEGFR) pathway, and AZD 2171 (Astra Zeneca), targeting VEGFR. The Met-selective inhibitor PF02341066 (originally developed by Pfizer) has been made available through the NCI Division of Cancer Treatment and Diagnosis. In addition to the assays already mentioned, RCC cell lines with and without mediator-targeted shRNA knockdowns or treatment with pharmacological pathway inhibitors will be compared for global changes in gene expression in collaboration with the Microarray Facility at the Advanced Technology Center, NCI. 3. Identification of novel HIF-2 inhibitors. Prior studies have shown that normoxic stabilization of HIF1-alpha alone, while capable of mimicking some aspects of VHL loss, are not sufficient to reproduce tumorigenesis, indicating that it is not the most critical oncogenic substrate of VHL. To isolate compounds that selectively modulate HIF2 for use as research tools and drug development leads, a cell-based high throughput screening assay of the NCI Natural Products Repository was developed in collaboration with CCRs Molecular Target Development Program (MTDP, Drs. James McMahon, Tawnya McKee and Girma Woldemichael). A series of luciferase reporter based reporter plasmids were designed and tested in the 786-0 RCC cell line leading to the selection of a single plasmid for optimization and screening. High throughput screening of the Natural Products Library yielded lead compound mixtures from which single compounds were then chromatographically separated into component structures for individual testing. Approximately 10 structures have been identified that have sub-100 micromolar IC50 values for inhibition of HIF2 reporter activity and minimal cell toxicity. These compounds are presently undergoing analysis in cell based assays for inhibition of HIF2-responsive genes such as VEGF.
