von Hippel-Lindau-Dependent Patterns of RNA Polymerase II Hydroxylation in Human Renal Clear Cell Carcinomas

We have previously shown that von Hippel-Lindau (VHL) regulates ubiquitylation and proline 1465 hydroxylation of the large subunit of RNA polymerase II, Rpb1, in human renal clear cell carcinoma (RCC) cell lines. Here, our goal was to determine the effect of this VHL function and the status of P1465 hydroxylation in human RCC tumors. Primary human tumors and matched normal kidney samples were probed for expression levels of the large subunit of RNA polymerase II (Rpb1), Rpb1 hydroxylated on P1465 [Rpb1(OH)], Rpb1 phosphorylated on Ser5 [Rpb1(S5P)], and proline hydroxylases PHD1, PHD2, and PHD3. Results from RCC tumors were categorized according to the status of VHL gene. Mechanistic analysis was performed in orthotopic xenograft model using 786-O RCC cells with wild-type (WT) VHL and knockdown of PHD2, characterized by high levels of Rpb1(OH) and PHD1. Levels of Rpb1(OH), PHD1, and PHD2 were significantly higher in RCC tumors compared with normal kidneys. RCC tumors with WT VHL had higher levels of Rpb1(OH) and PHD1 and lower levels of PHD2 than tumors with VHL gene alterations. Levels of Rpb1(OH) significantly correlated with levels of PHD1 in tumors and normal kidneys. Knockdown of PHD2 in 786-O VHL(+) cells resulted in a more malignant phenotype in orthotopic xenografts and higher expression of specific cell cycle regulators (CDC25A, cyclin-dependent kinase 2, CCNA2) compared with VHL(-) RCC cells. Elevated PHD1 concomitant with decreased PHD2 are causatively related to Rpb1 hydroxylation and oncogenesis in human RCC tumors with WT VHL gene. Thus, P1465-hydroxylated Rpb1 and PHD1 represent attractive drug targets for new RCC treatments.