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

Abstract

Purpose: 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.Experimental Design: 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. Clin Cancer Res; 16(21); 5142-52. ©2010 AACR.Renal clear cell carcinoma (RCC) is the most prevalent and malignant histologic type of kidney cancer. The majority of RCC (60-80%) is associated with intragenic mutations or hypermethylation of the von Hippel-Lindau (VHL) tumor suppressor gene and loss of heterozygosity at the VHL locus (3p26). In VHL-deficient tumors, activation of metabolic, angiogenic, and survival pathways resulting from VHL loss is an early and critical event in RCC tumorigenesis. In cases of RCC with the wild-type (WT) VHL gene, oncogenesis is not well understood. That is, it is not clear which activities of VHL are retained in those tumors.VHL is a substrate recognition molecule of an E3 ubiquitin ligase complex, recognizing hydroxylation of proline within the LxxLAP motif, which leads to ubiquitylation of the α subunits of hypoxia-inducible transcription factor (HIF; ref. 1). This hydroxylation is mediated by two of the O 2 -, Fe(II)-, and oxyglutarate-regulated Egln9-type proline hydroxylases, PHD2 and PHD3 (2). The function of the third enzyme from this group, PHD1, is less well understood, but it is known to hydroxylate conserved LQYLAP motifs of IKKβ and, by doing so, inhibit activation of the NF-κB transcription factor (3).Importantly, we have discovered that VHL mediates ubiquitylation of the l...

Von Hippel-Lindau Disease: Genetics and Role of Genetic Counseling in a Multiple Neoplasia Syndrome

“…[29][30][31] Furthermore, genetic instability in tumors is driven by VHL loss. 32 pVHL has been shown to mediate transcriptional regulation of nuclear factor-kB, 33 Rbp1 large subunit of RNA polymerase complex II, 34,35 p53 tumor suppressor, 36 p400 chromatin remodeling factor, 37 and JunB transcription factor via protein kinase C 25,38 ; however, pVHL also plays an important transcription-independent role in the regulation of extracellular matrix (ECM) and the microtubule cytoskeleton. pVHL is involved in the correct formation and turnover of the ECM 39 by interacting with collagen IV 40 and fibronectin.…”

TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma

“…Human VHL(−) and VHL(+) 786-O, A498 and Caki-1 cells were previously described (Mikhaylova et a., 2008, Yi et al, 2010). All cell lines are periodically authenticated (Genetica, Cincinnati, OH).…”

“…Fresh-frozen samples or paraffin-embedded samples of ccRCC and matched normal kidney were obtained and analyzed for VHL gene sequence and protein expression as described previously (Yi et al, 2010). All human samples were anonymous and exempt from IRB protocol.…”

“…All human samples were anonymous and exempt from IRB protocol. Lysates were analyzed for proteins of interest using semi-quantitative immunoblotting (Yi et al, 2010, Mikhaylova et al, 2012). …”

The role of HIF1α in renal cell carcinoma tumorigenesis

“…Currently, murine xenograft models using human RCC-derived cell lines or human ccRCC explants are used to test new drugs for efficacy (e.g. [42–46]).…”