Department of Pathology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center, PO Box 616, 6200 MD Maastricht, The Netherlands.
Am J Pathol. 2010 Feb;176(2):575-84. doi: 10.2353/ajpath.2010.090442. Epub 2009 Dec 30.
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.
Gremlin1 (GREM1) 是一种骨形态发生蛋白拮抗剂和潜在的血管生成调节基因，其启动子的异常高甲基化可导致人类恶性肿瘤中该基因沉默。在此，我们研究了在透明细胞肾细胞癌 (ccRCC) 中 GREM1 的甲基化情况及其对肿瘤特征和临床结果的影响。通过甲基化特异性 PCR 和/或 bisulfite 测序分析了 ccRCC 细胞系和两个独立患者系列的 ccRCC 中 GREM1 启动子的三个 CpG 岛区域（i、ii 和 iii）。将结果与临床病理和血管生成参数相关联。ccRCC 细胞系的 bisulfite 测序显示 GREM1 甲基化与 GREM1 mRNA 的缺失相关。ccRCC 中 GREM1 甲基化的发生率因区域而异：区域 i、ii 和 iii 的发生率分别为 55%、24%和 20%。在基于医院的 ccRCC 系列（n = 150）中，区域 iii 的 GREM1 甲基化与肿瘤大小增加（P = 0.02）、分期（P = 0.013）、分级（P = 0.04）、肿瘤（P = 0.001）和内皮细胞（P = 0.0001）增殖有关，与平均血管密度降低（P = 0.001）有关。在单变量分析中，与非甲基化 ccRCC 相比，区域 iii 甲基化的 ccRCC 患者的生存明显较差（风险比 [HR] = 2.35，95%置信区间 [CI]：1.29 至 4.28），但在多变量分析中则不然（HR = 0.88，95% CI：0.45 至 1.74）。在基于人群的验证系列（n = 185）中，区域 iii 的 GREM1 甲基化与 Fuhrman 分级增加（P = 0.03）和总生存期缩短相关（P = 0.001），无论是在单变量还是多变量分析中（HR = 2.32，95% CI：1.52 至 3.53 和 HR = 2.27，95% CI：1.44 至 3.59）。区域 iii 启动子甲基化与恶性程度增加之间的强相关性及其与活跃血管生成的相关性表明，GREM1 在 ccRCC 发生和肿瘤血管生成中起作用。
