Suppressive effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation

Increasing data implicate iron accumulation in tumorigenesis of the kidney, particularly the clear cell renal cell carcinoma (ccRCC) subtype. The von Hippel Lindau (VHL)/hypoxia inducible factor-alpha (HIF-alpha) axis is uniquely dysregulated in ccRCC and is a major regulator and regulatory target of iron metabolism, yet the role of iron in ccRCC tumorigenesis and its potential interplay with VHL inactivation remains unclear. We investigated whether ccRCC iron accumulation occurs due to increased cell dependency on iron for growth and survival as a result of VHL inactivation. Free iron levels were compared between four VHL-mutant ccRCC cell lines (786-0, A704, 769-P, RCC4) and two benign renal tubule epithelial cell lines (RPTEC, HRCEp) using the Phen Green SK fluorescent iron stain. Intracellular iron deprivation was achieved using two clinical iron chelator drugs, deferasirox (DFX) and deferoxamine (DFO), and chelator effects were measured on cell line growth, cell cycle phase, apoptosis, HIF-1 alpha and HIF-2 alpha protein levels and HIF-alpha transcriptional activity based on expression of target genes CA9, OCT4/POU5F1 and PDGF beta/PDGFB. Similar assays were performed in VHL-mutant ccRCC cells with and without ectopic wild-type VHL expression. Baseline free iron levels were significantly higher in ccRCC cell lines than benign renal cell lines. DFX depleted cellular free iron more rapidly than DFO and led to greater growth suppression of ccRCC cell lines (> 90% at similar to 30-150 mu M) than benign renal cell lines (similar to 10-50% at up to 250 mu M). Similar growth responses were observed using DFO, with the exception that a prolonged treatment duration was necessary to deplete cellular iron adequately for differential growth suppression of the less susceptible A704 ccRCC cell line relative to benign renal cell lines. Apoptosis and G1-phase cell cycle arrest were identified as potential mechanisms of chelator growth suppression based on their induction in ccRCC cell lines but not benign renal cell lines. Iron chelation in ccRCC cells but not benign renal cells suppressed HIF-1 alpha and HIF-2 alpha protein levels and transcriptional activity, and the degree and timing of HIF-2 alpha suppression correlated with the onset of apoptosis. Restoration of wild-type VHL function in ccRCC cells was sufficient to prevent chelator-induced apoptosis and G1 cell cycle arrest, indicating that ccRCC susceptibility to iron deprivation is VHL inactivation-dependent. In conclusion, ccRCC cells are characterized by high free iron levels and a cancer-specific dependency on iron for HIF-alpha overexpression, cell cycle progression and apoptotic escape. This iron dependency is introduced by VHL inactivation, revealing a novel interplay between VHL/HIF-alpha dysregulation and ccRCC iron metabolism. Future study is warranted to determine if iron deprivation using chelator drugs provides an effective therapeutic strategy for targeting HIF-2 alpha and suppressing tumor progression in ccRCC patients.