Abstract
The tumor suppressor gene, Von Hippel-Lindau (VHL), is frequently mutated in the most common form of kidney cancer, clear cell renal cell carcinoma (CCRCC). In hypoxic conditions, or when there is a VHL mutation, the hypoxia inducible factors, HIF1α and HIF2α, are stabilized and transcribe a panel of genes associated with cancer such as vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor (PDGF), and glucose transporter 1 (GLUT1). Recent studies in clear cell kidney cancer have suggested that HIF2α, but not HIF1α, is the critical oncoprotein in the VHL pathway. Therefore, targeting HIF2α could provide a potential therapeutic approach for patients with advanced CCRCC. Since iron regulatory protein 1 (IRP1) is known to inhibit the translation of HIF2α, we investigated whether Tempol, a stable nitroxide that activates IRP1 towards IRE-binding, might have a therapeutic effect on a panel of human CCRCC cells expressing both HIF1α and HIF2α. We first evaluated the protein expression of HIF1α and HIF2α in 15 different clear cell renal carcinoma cell lines established from patient tumors in our laboratory. Tempol decreased the expression of HIF2α, and its downstream targets in all the cell lines of the panel. This effect was attributed to a dramatic increase of IRE-binding activity of IRP1. Several cell lines were found to have an increased IRP1 basal activity at 20% O2 compared to 5% O2, which may lower HIF2α expression in some of the cell lines in a VHL-independent manner. Taken together our data identify Tempol as an agent with potential therapeutic activity targeting expression of HIF2α in VHL-deficient clear cell kidney cancer and illustrate the importance of studying biochemical processes at relevant physiological O2 levels.
Highlights
In the United States, an estimated 64,770 new cases of kidney cancers will be diagnosed in 2012 and about 13,570 people will die of this disease [1]
In this report, using a panel of Von Hippel-Lindau (VHL)-deficient clear cell renal cell carcinoma (CCRCC) cell lines that express increased either HIF1α or HIF2α or both, we investigated whether Tempol inhibits the translation of HIF2α by inducing the iron-responsive element (IRE) binding activity of iron regulatory protein 1 (IRP1) and whether Tempol might be a potential targeted therapy for CCRCC
Using 30μg of nuclear extracts and similar to that observed in the previous experiment, Tempol significantly decreased the level of nuclear HIF2α, suggesting that its transcriptional activity was compromised
Summary
In the United States, an estimated 64,770 new cases of kidney cancers will be diagnosed in 2012 and about 13,570 people will die of this disease [1]. The most common type of kidney cancers, clear cell renal cell carcinoma (CCRCC), is associated with mutations of the VHL gene [2,3,4]. The product of the VHL gene is associated with ubiquitination and degradation of the hypoxia inducible factor (HIF) through an oxygen-sensing mechanism [5,6]. HIF1α and HIF2α are hydroxylated by prolyl-hydroxylases in an iron-dependent manner. This post-translational modification allows recognition of HIF by the VHL complex and leads to its degradation by the proteasome [7,8]. In the absence of oxygen, or in the www.impactjournals.com/oncotarget
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