Hypoxia-inducible factor (HIF) plays an important role in renal tumourigenesis. In the majority of clear cell RCC (ccRCC), the most frequent and highly vascularized RCC subtype, HIF is constitutively activated by inactivation of the von Hippel-Lindau gene. Of the HIF subunits, HIF-2alpha appears to be more oncogenic than HIF-1alpha, in that HIF-2alpha activates pro-tumourigenic target genes. In addition, recent studies indicate that HIF-1alpha, more than HIF-2alpha, can undergo proteasomal degradation in VHL - /- RCC cells. A more detailed understanding of the molecular basis of hypoxia and angiogenesis in renal carcinogenesis has set the stage for the development of targeted therapies, inhibiting multiple HIF-related pathways, such as the phosphatidylinositol 3-kinase-AKT-mTOR, RAS/RAF/MAP, and VEGF signalling routes. However, despite the positive results of these targeting agents in progression-free survival, clinical resistance remains an issue. Recent pre-clinical studies have suggested new targeting approaches such as inhibition of HIF-driven key metabolic enzymes and have introduced new HIF targeting agents, such as histone deacetylase inhibitors, with successful anti-neoplastic effects. In this review, we discuss existing and novel findings about RCC carcinogenesis, with subsequent clinical implications.

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The genetic and epigenetic alterations that underlie cancer pathogenesis are rapidly being identified. This provides novel insights in tumor biology as well as in potential cancer biomarkers. The somatic mutations in cancer genes that have been implemented in clinical practice are well defined and very specific. For epigenetic alterations, and more specifically aberrant methylation of promoter CpG islands, evidence is emerging that these markers could be used for the early detection of cancer as well as prediction of prognosis and response to therapy. However, the exact location of biologically and clinically relevant hypermethylation has not been identified for the majority of methylation markers. The most widely used approaches to analyze DNA methylation are based on primer-and probe-based assays that provide information for a limited number of CpG dinucleotides and thus for only part of the information available in a given CpG island. Validation of the current data and implementation of hypermethylation markers in clinical practice require a more comprehensive and critical evaluation of DNA methylation and limitations of the techniques currently used in methylation marker research. Here, we discuss the emerging evidence on the importance of the location of CpG dinucleotide hypermethylation in relation to gene expression and associations with clinicopathologic characteristics in cancer. Clin Cancer Res; 17(13); 4225-31. Ó2011 AACR.

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P ‫؍‬ 0.02), stage (P ‫؍‬ 0.013), grade (P ‫؍‬ 0.04), tumor (P ‫؍‬ 0.001), and endothelial cell (P ‫؍‬ 0.0001) proliferation and decreased mean vessel density (P ‫؍‬ 0.001) in a hospital-based ccRCC series (n ‫؍‬ 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] ‫؍‬ Clear cell renal cell carcinoma (ccRCC) accounts for Ϸ75% of all cases of renal cell cancer, 1 and is characterized by increased vascularization and an unclear clinical prognosis. Currently, patient performance status, tumor size, nodal and distant metastasis (TNM)-stage, and Fuhrman nuclear grade are the most useful predictors of patient outcome.2 However, interest in additional prognostic molecular markers is growing. Inactivation of the von Hippel-Lindau (VHL) gene has been shown to be a common and early event in the carcinogenesis of ccRCC.3-5 Fifty to 70% of ccRCC tumors harbor a VHL mutation, 5,6 and in 5% to 20% of ccRCCs VHL is silenced by promoter CpG island hypermethylation. 7,8 Although defective VHL functioning is a key event in the development in both sporadic and hereditary ccRCCs, alterations in the structure or regulation of the VHL gene do not Supported by the Kootstra-Talent Fellowship Program, Maastricht

Purpose In this era of molecular diagnostics, prediction of clear-cell renal cell cancer (ccRCC) survival requires optimization, as current prognostic markers fail to determine individual patient outcome. Epigenetic events are promising molecular markers. Promoter CpG island methylation of cysteine dioxygenase type 1 (CDO1), which was identified as prognostic marker for breast cancer, is studied as a potential marker for ccRCC survival. Experimental Design We collected primary tissues of 365 ccRCC cases identified within the prospective Netherlands Cohort Study (NLCS). In this population-based series, CDO1 promoter methylation was observed in 124 of 324 (38.3%) patients with successful methylation-specific PCR analysis. Kaplan-Meier curves and Wilcoxon tests were used to evaluate 10-year ccRCC-specific survival. Cox regression analysis was used to obtain crude and multivariate HRs and 95% confidence intervals (CI). The relative prognostic value of multivariate models with and without CDO1 promoter methylation was compared using likelihood-ratio tests. Results Patients with CDO1 promoter methylation have a significantly poorer survival than those without (Wilcoxon P = 0.006). Differences in survival were independent of other prognostic factors, including age and sex (HR, 1.66; 95% CI, 1.12–2.45) and TNM stage, tumor size, and Fuhrman grade (HR, 1.89; 95% CI, 1.25–2.85). Multivariate models performed better with than without CDO1 promoter methylation status (likelihood-ratio P = 0.003). Survival curves were validated in an independent series of 280 ccRCC cases from The Cancer Genome Atlas (TCGA; Wilcoxon P< 0.001). Conclusions CDO1 promoter methylation may not substitute common prognostic makers to predict ccRCC survival, but offers additional, relevant prognostic information, indicating that it might be a novel molecular marker to determine ccRCC prognosis.