Abstract 804: Regulation of E2F1 by pVHL predicts survival in RCC patients

Abstract

Abstract Inactivation of VHL is the most common cause of Clear Cell Renal Cell Carcinoma (CCRCC) and is associated with the stabilization of Hypoxia Inducible Factor (HIF)-alpha subunits. When reconstituting several VHL-deficient RCC cell lines with pVHL, we observed markedly decreased mRNA and protein expression of the E2F1 transcription factor. Reporter assays measuring endogenous E2F1 transcriptional activity similarly responded to exogenous pVHL expression in a dose-dependent manner. From a panel of stable RCC cell lines reconstituted with disease-associated allelic variants of VHL, we demonstrate that all alleles tested downregulate E2F1 expression irregardless of their ability to regulate HIF1-alpha, suggesting a novel HIF-independent function for pVHL. Accordingly, RNAi knockdown of HIF-alpha in RCC cells does not alter E2F1 expression, whereas RNAi knockdown of VHL does. To investigate patho-physiological consequences of VHL-loss on E2F1 in vivo, we confirmed that zebrafish embryos lacking a VHL ortholog exhibit upregulation of E2F1 target genes mcm3 and mcm5. Blocking protein synthesis with cycloheximide in RCC cells with or without pVHL did not affect E2F1 protein turnover. To examine the clinical implication of pVHL-E2F1 interplay, we performed immunohistochemical analyses of 140 RCC patients and compared tumor tissue to normal kidney tissue from the same patients. Patients included in this study underwent nephrectomy for primary RCC. Clinical and histological characteristics together with follow-up were collected from medical records. Eight patients were known von Hippel-Lindau patients and expressed significantly higher nuclear levels of E2F1 compared to patients with Clear Cell (CC, N=107) or non-CC histology (N=25) (Student's T-test, P<0.05 both). Small tumors (diameter<7 cm according to the AJCC grading system) showed significantly higher nuclear E2F1 expression compared to larger tumors (P=0.007), suggesting prediction for survival. Cox regression analysis for 5-year survival showed significant association between E2F1 (continuous variable) and disease-free survival (DFS) as well as for overall survival (OS) (Log Likelihood Ratio test; P=0.02 and P=0.01 respectively). This correlation demonstrates that low nuclear expression of E2F1 is a risk factor for poor survival in RCC patients. Using 20% nuclear E2F1 expression as cut-off point patients could be categorized into low (<20%) or high (≥20%) risk groups. DFS and OS were accurately depicted in Kaplan-Meier curves (Log Rank test, P=0.02 and P=0.002, respectively). In conclusion, nuclear E2F1 expression is regulated by VHL and predictive for survival in RCC patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 804.