Genomic Medicine Institute, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Genes Chromosomes Cancer. 2011 Aug;50(8):654-61. doi: 10.1002/gcc.20887. Epub 2011 May 16.
We recently identified germline methylation of KILLIN, a novel p53-regulated tumor suppressor proximal to PTEN, in >1/3 Cowden or Cowden syndrome-like (CS/CSL) individuals who are PTEN mutation negative. Individuals with germline KILLIN methylation had increased risks of renal cell carcinoma (RCC) over those with PTEN mutations. Therefore, we tested the hypothesis that KILLIN may be a RCC susceptibility gene, silenced by germline methylation. We found germline hypermethylation by combined bisulfite restriction analysis in at least one of the four CpG-rich regions in 23/41 (56%) RCC patients compared to 0/50 controls (P < 0.0001). Of the 23, 11 (48%) demonstrated methylation in the -598 to -890 bp region in respect to the KILLIN transcription start site. Furthermore, 19 of 20 advanced RCC showed somatic hypermethylation upstream of KILLIN, with the majority hypermethylated at more than one CpG island (13/19 vs. 3/23 with germline methylation, P < 0.0001). qRT-PCR revealed that methylation significantly downregulates KILLIN expression (P = 0.05), and demethylation treatment by 5-aza-2'deoxycytidine significantly increased KILLIN expression in all RCC cell lines while only increasing PTEN expression in one line. Furthermore, targeted in vitro methylation revealed a significant decrease in KILLIN promoter activity only. These data reveal differential epigenetic regulation by DNA promoter methylation of this bidirectional promoter. In summary, we have identified KILLIN as a potential novel cancer predisposition gene for nonsyndromic clear-cell RCC, and the epigenetic mechanism of KILLIN inactivation in both the germline and somatic setting suggests the potential for treatment with demethylating agents.
我们最近发现，一种新型 p53 调控的抑癌基因 KILLIN 在超过 1/3 的 PTEN 阴性 Cowden 或 Cowden 综合征样（CS/CSL）个体中存在胚系甲基化，而 PTEN 突变阴性。具有胚系 KILLIN 甲基化的个体发生肾细胞癌（RCC）的风险高于具有 PTEN 突变的个体。因此，我们测试了 KILLIN 可能是一个 RCC 易感基因的假说，该基因被胚系甲基化沉默。我们发现，与 50 名对照（P < 0.0001）相比，至少在 41 名 RCC 患者的四个 CpG 丰富区域中的一个区域中，通过联合亚硫酸氢盐限制性分析检测到胚系高甲基化。在 23 名患者中，有 11 名（48%）在 KILLIN 转录起始位点的-598 至-890 bp 区域显示甲基化。此外，20 名晚期 RCC 中有 19 名显示 KILLIN 上游的体细胞高甲基化，大多数在一个以上 CpG 岛中高甲基化（13/19 与胚系甲基化相比，P < 0.0001）。qRT-PCR 显示，甲基化显著下调 KILLIN 的表达（P = 0.05），用 5-氮杂-2'-脱氧胞苷进行去甲基化处理显著增加了所有 RCC 细胞系中的 KILLIN 表达，而仅在一条线中增加了 PTEN 表达。此外，靶向体外甲基化仅显示出 KILLIN 启动子活性的显著降低。这些数据揭示了该双向启动子的 DNA 启动子甲基化对其进行的差异表观遗传调控。总之，我们已经确定 KILLIN 是一种潜在的新的非综合征性透明细胞 RCC 易感性基因，KILLIN 在胚系和体细胞中的失活的表观遗传机制表明，用去甲基化剂进行治疗具有潜力。
