Abstract 2391: Hsp90-Dependent Regulation of HIF and angiogenic potential in clear-cell renal cell carcinoma

Abstract

Abstract Tumor vascularization is a critical component of cancer progression, malignancy, and metastasis and hypoxia inducible factor (HIF) is a key regulator of these processes. HIF transcriptional activity regulates numerous pro-angiogenic cytokines that act upon cancer cells and the surrounding tumor stroma to cooperatively stimulate tumor vascularization. HIF is constitutively activated in clear cell renal cell carcinoma (CCRCC), the most lethal and angiogenic type of kidney cancer, due to genetic or functional loss of the HIF-targeting VHL ubiquitin ligase protein. In the absence of VHL-dependent HIF degradation, pharmacologic targeting of HIF may be exploited as a means to suppress HIF-dependent signaling. HIF is also a client protein for Hsp90, a molecular chaperone regulating a number of substrates that support cancer progression. Hsp90 inhibitors possess anti-tumorigenic and anti-angiogenic properties, due in part to their ability to inhibit HIF function. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in CCRCC and other solid tumors, highlighting the need to further evaluate the role of Hsp90 in CCRCC angiogenic processes and HIF signaling. Approach: We compared the molecular and functional effects of 3 Hsp90 inhibitors, using VHL replaced cells as HIF suppressed controls. We examined the prototypic Hsp90 inhibitor 17-AAG, a newer, more potent totally synthetic small molecule derivative (EC154), and LBH589, an HDAC inhibitor in clinical use that also impairs Hsp90 function. Our assays consisted of analysis of drug effects on 1) genome wide HIF ChIP and RT-PCR 2) cytokine profiles, 3) endothelial cell tubule assays, and 4) limited phospho-proteomic analysis of signaling proteins. Results: These inhibitors exhibit variable suppression of HIF target genes, illustrating the complexity associated with these pathways. Cytokine arrays indicated that while VEGF expression decreased in response to all drugs, a number of proteins unexpectedly increased. In addition, these changes did not always correlate with VHL-dependent HIF suppression. Although Hsp90 inhibitors initiate the destruction of a number of proteins and impact upon numerous signaling cascades, the protein-centric effects of Hsp90 inhibition in cancer cells remain unknown. To explore this, we utilized a commercial phosphoproteome array to examine the effects of Hsp90 inhibition upon the activation status of over 1300 key signaling proteins. Finally, to understand how alterations in these signaling profiles modulate angiogenic potential, conditioned medium from untreated cells, VHL replaced cells, or drug treated cells was evaluated in a tubule assay. Collectively, the results of these studies will further define the role of HIF in CCRCC tumor angiogenesis, add to our understanding of molecular events that contribute to clinical failure of Hsp90 inhibitors, and shed light on alternative approaches for CCRCC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2391.