von Hippel-Lindau-dependent patterns of RNA polymerase II hydroxylation in human renal clear cell carcinomas.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yi
Ying
Y
Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521, USA.
Mikhaylova
Olga
O
Mamedova
Aygun
A
Bastola
Prabhat
P
Biesiada
Jacek
J
Alshaikh
Enas
E
Levin
Linda
L
Sheridan
Rachel M
RM
Meller
Jarek
J
Czyzyk-Krzeska
Maria F
MF
D000818:Animals; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006900:Hydroxylation / Q000235:genetics; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational; D012319:RNA Polymerase II / Q000378:metabolism*; D014183:Transplantation, Heterologous; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D009687:Nuclear Proteins; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C447780:EGLN2 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D012319:RNA Polymerase II