Prognostic significance of Gremlin1 (GREM1) promoter CpG island hypermethylation in clear cell renal cell carcinoma

Abstract

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] = 2.35, 95% confidence interval [CI]:1.29 to 4.28), but not in multivariate analysis (HR = 0.88, 95% CI: 0.45 to 1.74). In a population-based validation series (n = 185), GREM1 region iii methylation was associated with increased Fuhrman grade (P = 0.03) and decreased overall survival (P = 0.001) in univariate and multivariate analysis (HR = 2.32, 95% CI: 1.52 to 3.53 and HR = 2.27, 95% CI: 1.44 to 3.59, respectively). The strong correlation between GREM1 region iii promoter methylation and increased malignancy and its correlation with active angiogenesis indicates a role for GREM1 in ccRCC carcinogenesis and tumor angiogenesis.

Figure 1

GREM1 promoter CpG island, methylation analyses, and mRNA expression in ccRCC cell lines. GREM1 putative promoter CpG island relative to the transcription start site (TSS), including the locations of BSseq primer sets (i, ii, TSS, and iii; grey arrows: sequence primer locations) and MSP primer sets i, ii, and iii (underlined: flank PCR primer locations, black arrows: methylation specific PCR primer location, respectively) (A) with representive BSseq (B), MSP, asterisk represents clones for bisulfite sequencing of the cell lines SKRC1, SKRC10, SKRC52, and SKRC59 for region i, ii, TSS and iii. (C), and mRNA expression (D) results of ccRCC cell lines SKRC1, SKRC10, SKRC52, SKRC59. mRNA expression data resulted after three independent experiments. Open/closed squares indicate unmethylated/methylated CpGs. U indicates unmethylated product; M, methylated product; IVD, methylation positive control; HUV, methylation negative control; H2O(1), Flank PCR control without DNA; H2O(2), MSP control without DNA; DAC, 5′aza-2-deoxycytidine; TSA, Trichostatin A.

Figure 2

Overall cause-specific survival curves for ccRCC patients according to GREM1 promoter CpG island methylation at region i (A), ii (B), and iii (C and D), with A, B, and C for patients of the hospital-based series and D for patients of the population-based series.

Figure 3

Prognostic significance of GREM1 promoter CpG island methylation in hospital-based and population-based ccRCC patient series.