Presentation Authors: Thomas Siff*, Christopher Greene, Nikita Sharma, Peter Fiorica, Arun Menon, Gary Smith, Kenneth Gross, Eric Kauffman, Buffalo, NY
Introduction: The main dysregulated pathway of clear cell renal cell carcinoma (ccRCC), the von Hippel Lindau (VHL)/hypoxia inducible factor-Î± (HIF-Î±) axis, is a key regulator and regulatory target of iron metabolism, yet the role of iron uptake in ccRCC tumorigenesis is unclear. Iron promotes HIF-2Î± translation, and iron overexposure induces ccRCC tumors in rodents and is associated with increased diagnostic risk of RCC in humans. We previously reported that the tumor level of the primary iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71), is an independent predictor of worse metastasis-free survival in ccRCC patients. Here we tested the hypothesis that TfR1 directly mediates key tumorigenic properties of ccRCC cells
Methods: Baseline TfR1 protein and RNA levels were measured using Western blot and RT-PCR in 4 ccRCC and 2 benign renal epithelial cell lines. Stable TfR1 protein knockdown (KD) in the ccRCC cell line, 786-0, was performed using two shRNA expression vectors (KD1, KD2) and an empty vector control (EV). 786-0-KD1, 786-0-KD2 and 786-0-EV cell lines were compared with regards to several cell measures, including cell proliferation via MTT assays; apoptosis rates via flow cytometry quantification of Annexin V stain; cell cycle phase distribution via flow cytometry quantification of Propidium Iodide stain; HIF-2Î± protein levels via Western blot; and HIF-2Î± transcriptional activity via RT-PCR quantification of the Oct4 (HIF-2Î± target) gene transcript.
Results: TfR1 protein and RNA levels were significantly higher in ccRCC cell lines relative to benign renal epithelial cell lines. Approximately 90% and 99% reductions in TfR1 protein level were achieved in 786-0-KD1 and 786-0-KD2 cell lines, respectively, relative to 786-0-EV cells. 786-0-KD1 and 786-0-KD2 cells had significant reductions relative to 786-0-EV cells in HIF-2Î± protein level, HIF-2Î± transcriptional activity and cell proliferation. The degree of HIF-2Î± suppression and growth suppression correlated with the degree of TfR1 knockdown. TfR1 knockdown significantly reduced the proportion of 786-0 cells in the S cell cycle phase, and was associated with non-significant increases in apoptosis.
Conclusions: The primary iron uptake receptor, TfR1, plays a critical role in HIF-2Î± overexpression and proliferation of ccRCC cells. Additional research is warranted to determine whether targeting of TfR1 protein and cellular iron uptake provides an effective clinical strategy for suppressing HIF-2Î± and tumor growth in ccRCC patients.