Regulation of Erythropoietin Activity in Clear Renal Cell Carcinoma

Clear-cell renal cell carcinoma (ccRCC) is associated with the mutated von Hippel-Lindau (VHL) gene leading to the activation of hypoxia-inducible factor 1A (HIF1A) and subsequent overexpression of erythropoietin (EPO). We analyzed tumor and healthy tissues from 43 ccRCC patients after radical nephrectomy and cultured 786-O (biallelic <i>VHL</i> inactivation) and Caki-1 (wild-type <i>VHL</i>) cells in normal (21% O₂) and low oxygen (3% O₂) with 10% and 2% fetal bovine serum (FBS). DNA sequencing, including Sanger sequencing, MLPA and LOH, revealed 27 somatic mutations of <i>VHL</i> in ccRCC. HIF1A protein showed decreased or no expression in tumors compared to healthy tissue, independent of <i>VHL</i> alteration. The 786-O cells showed increased HIF1A protein expression after 48 h under low oxygen and 10% FBS. EPO and erythropoietin receptor (EPOR) were significantly decreased in ccRCC without HIF1A expression. EPO mRNA increased in the 786-O cells at 3% O₂ after 48 h, while the Caki-1 cells had low or no EPO expression. Hypoxia increased EPOR mRNA in the Caki-1 cells at 10% FBS, but decreased in the 786-O cells at 2% FBS after 48 h. JAK2/STAT5A activity was increased only in HIF1A-positive tumors. These results suggest that EPO/EPOR activation in ccRCC is mainly driven by low oxygen, not VHL regulation of hypoxia-related responses.