Login to Access Video or Poster Abstract: MP60-02
Sources of Funding: This project was supported by the Roswell Park Cancer Institute Alliance Foundation.

Introduction

The von Hippel Lindau (VHL)/hypoxia inducible factor-alpha (HIF-α) pathway is commonly dysregulated in clear cell renal cell carcinoma (ccRCC) and potentially exacerbated by oxidative stress. Ferric iron is the most abundant heavy metal in the human body and is required for nephrogenesis in utero; it is also a major source of intracellular oxidative stress capable of inducing ccRCC tumorigenesis in rodent models. In addition to its known requirement for DNA synthesis, iron was recently shown to stabilize HIF2-α transcript by inhibiting iron regulatory protein 1. Still, a role for iron in human ccRCC tumorigenesis remains largely unexplored. Here we test the hypotheses that intracellular iron levels are increased in ccRCC cells and that iron reduction can effectively reduce HIF2-α expression and suppress ccRCC cell growth.

Methods

Iron levels were measured in 100 RCC patient tumors using Prussian Blue stain and compared to levels in benign renal tubule epithelium. Protein levels of the main iron uptake protein, transferrin receptor 1 (TfR1/CD71), were compared between 4 VHL-deficient ccRCC cell lines and 2 benign renal epithelial cell lines by flow cytometry and western blot. Micromolar concentrations of 3 clinically approved iron chelator drugs, deferoxamine (DFO), deferiprone (DFP), and deferisirox (DFX) were tested by MTT assay for effects on ccRCC versus benign renal cell proliferation. Cell viability and apoptosis induction with DFO treatment was measured in select ccRCC cell lines using flow cytometry analysis of annexin-V and 7-AAD stains, as were effects of DFO on ccRCC HIF2-α protein levels.

Results

Increased intracellular iron was detected in ccRCC tumors compared to benign renal tubule epithelium. All ccRCC cell lines had elevated TfR1 expression compared to benign renal cell lines. Iron chelation treatment using DFO, DFP, or DFX achieved 80-90% growth reduction in all ccRCC cell lines at clinically relevant concentrations, while benign renal cell lines were relatively resistant. The mechanism underlying ccRCC growth inhibition included cell death via apoptosis, whereas benign renal cells had no increased death even after prolonged iron chelation treatment. Intriguingly, iron chelation in ccRCC cell lines effectively downregulated HIF2-α in a time- and concentration-dependent manner.

Conclusions

These data indicate that intracellular increases in iron content occur during human ccRCC tumorigenesis and support iron chelation as a potential therapeutic strategy for targeting HIF2-α and inducing cancer cell death in ccRCC patients.

Funding

This project was supported by the Roswell Park Cancer Institute Alliance Foundation.