1. Define the integration of growth factor receptor pathway components with other oncogenic signaling pathways in clear cell 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. 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 and oncogenic signaling observed in clear cell RCC. Prior studies under this goal 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. Current work under this aim focuses on the mechanistic basis of constitutive Met kinase activation that is prevalent in clear cell RCC. This work is subdivided into confirming and measuring the prevalence of dysregulated Met activation in human pVHL-negative clear cell RCC tumor specimens, and identifying the molecular basis for constitutive Met activation using tumor derived cell lines. 2. Preclinical assessment of inhibitors of the VHL/HIF/mTor axis as research tools and to assess their preclinical efficacy. Current work toward this goal utilizes agents targeting components of the VHL/HIF signaling axis and angiogenic pathways, such as the experimental drug candidates ZD6474, targeting both the EGFR and vascular endothelial growth factor receptor (VEGFR) pathways, AZD2171, targeting VEGFR, and the PDK1 inhibitor AR12 (OSU-03012) recently obtained from Arno Therapeutics, to interrogate the integration of growth factor pathways with those driving metabolic pathways toward anaerobic glycolysis (the Warburg effect) in clear cell carcinoma as well as RCC tumors associated with BHD and HLRCC. PDK1 (3-phosphoinositide dependent protein kinase-1) is a critical regulator of Akt, PKC, S6K and SGK, with regulatory input from several growth factors, hormones, and HIF-regulated pathways affecting cell metabolism. These studies exploit genetically altered and engineered cultured cell models derived from UOB patient tumors, as well as transgenic and knock-out mouse models of these diseases also developed in UOB. 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 may not be the most critical oncogenic substrate of VHL protein. To isolate compounds that selectively modulate HIF2-alpha 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 Dr. Tawnya McKee in the Molecular Targets Laboratory (NCI/CCR/MTL). A series of reporter plasmids were designed that placed tandem copied of the minimal hypoxia response element of the VEGF gene in control of the gene encoding firefly luciferase. These plasmids were transfected into the VHL-and HIF1-negative RCC tumor-derived cell line 786-0 and derived cell lines were optimized for use in high-throughput detection systems in the MTL. Counter screens for global transcriptional repression as well as cell toxicity were components of the final screening strategy. Leads from screening of the NCI Natural Products Repository were chromatographically separated into component structures yielding approximately 40 pure compounds with micromolar or submicromolar IC50 values, greater than 80% inhibition and less than 5% cell toxicity. These hits were then re-screened for VEGF protein secretion by 786-0 in the UOB. A majority of the compounds identified in the initial screen showed also modulation of VEGF secretion;dose response curves of the 6 most active compounds revealed 2 compounds with low micromolar inhibition of VEGF protein secretion. These leads, as well as the 30 remaining most active hits are now being tested in PCR-based assays for their affects on a panel of 7 HIF-regulated genes in three clear cell RCC-derived cell lines: 786-0 (HIF2 expression only), RCC4 (equal expression of HIFs 1 and 2) and SK-RC-28 (engineered for suppression of HIF2). This evaluation should distinguish compounds with activity toward HIF1, 2 or both. Future biological characterization of these compounds will begin with biochemical confirmation of activity against the HIF2 promoter, as well as bioassays for proliferation, survival, motility and anchorage independent growth. Later animal studies will assess toxicity, PK and efficacy in tumor xenograft and UOB-developed mouse models of RCC.
