Carcinogenesis Advance Access originally published online on December 4, 2008
Carcinogenesis 2009 30(3):387-396; doi:10.1093/carcin/bgn275
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von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR
Institut National de la Santé et de la Recherche Médicale U727, Section of Renal Pharmacology and Physiopathology, School of Medicine, University Louis Pasteur, Strasbourg 67085, France
1 Department of Pathology, Hôpitaux Universitaires de Strasbourg, Strasbourg-Hautepierre 67200, France
2 Department of Urology, Hôpitaux Universitaires de Strasbourg, Strasbourg 67091, France
* To whom correspondence should be addressed. Tel: +333 90 24 34 56; Fax: +333 90 24 34 59; Email: thierry.massfelder{at}medecine.u-strasbg.fr
We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription–polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-β. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.
Abbreviations: BrdU, bromodeoxyuridine; cDNA, complementary DNA; CRCC, conventional renal cell carcinoma; FM, fentomolar; GAPDH, glyceraldehyde phosphate dehydrogenase; HIF, hypoxia-induced factor; mRNA, messenger RNA; PCR, polymerase chain reaction; PTHrP, parathyroid hormone-related protein; RCC, renal cell carcinoma; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TGF, tumor growth factor; UTR, untranslated region; UV, ultraviolet; VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau
Received September 3, 2008; revised November 28, 2008; accepted November 29, 2008.