Wnt Antagonist Gene Hypermethylation in Circulating DNA: Cancer Biomarker

Wnt通路的异常激活是许多类型的人类恶性肿瘤的共同特征。一些研究表明，编码分泌Wnt拮抗剂的基因在肿瘤细胞中经常被超甲基化沉默，这与这些蛋白通常作为肿瘤抑制因子的观点一致。分泌卷曲相关蛋白（sFRP）、Dickkopf （Dkk）和Wnt抑制因子（WIF）家族成员在肾细胞癌（RCC）中被高甲基化沉默。此外，恢复RCC细胞中sFRP-1的表达可显著抑制细胞增殖和肿瘤生长，并降低Wnt靶基因的表达，这表明sFRP-1的缺失是肾细胞癌变的关键事件。这些研究强烈表明，检测Wnt拮抗剂基因的高甲基化状态可以作为RCC的有用生物标志物。如果这些基因的高甲基化是RCC发展的早期事件，就像其他肿瘤所表明的那样，如果有可靠的、非侵入性的方法来识别基因的高甲基化，这将特别有价值。循环DNA中基因甲基化状态的评估已经被描述，这使得它有可能作为一种强大的癌症诊断筛查工具。然而，循环中的微量DNA和传统聚合酶链反应（PCR）方法的有限敏感性阻碍了这种诊断方式的发展。我们将利用实时PCR技术开发一种灵敏度和特异性更高的检测方法，以检测RCC患者血清样本中高度甲基化的Wnt拮抗剂基因。该项目将充分利用NCI社区内外合作者提供的碾压混凝土样本和专业知识。开发一种成功的检测Wnt拮抗剂基因超甲基化的方案，可以实现RCC的早期诊断和监测，也可能为癌症治疗的选择提供指导，所有这些都将对患者的生存产生深远的有益影响。在过去的一年中，各种技术问题引起了人们对用于检测高甲基化Wnt拮抗剂基因的PCR分析可靠性的关注。我们使用巢式PCR和实时PCR相结合的方法验证了编码sFRP-1、sFRP-4、sFRP-5和Dkk3基因对应的甲基化DNA的检测。然而，我们观察到大量不相关的PCR产物可能被解释为假阳性信号。通过包括PCR产物的熔化曲线分析，假阳性分配的可能性已经降低。基于对甲基化的sFRP-1 cDNA的研究，似乎可以通过省略嵌套PCR步骤，并依靠亚硫酸氢盐处理的甲基化与未甲基化DNA产生的PCR产物在熔融方面的固有差异，更好地区分真阳性和假阳性信号（Lorente等）。BMC癌症2008)。

Aberrant activation of Wnt pathways is a common feature of many types of human malignancies. Several studies have demonstrated that the genes encoding secreted Wnt antagonists are frequently silenced in tumor cells by hypermethylation, consistent with the idea that these proteins often function as tumor suppressors. Members of the secreted Frizzled-related protein (sFRP), Dickkopf (Dkk) and Wnt Inhibitory Factor (WIF) families are silenced by hypermethylation in renal cell carcinoma (RCC). Moreover, restoration of sFRP-1 expression in RCC cells markedly inhibited cell proliferation, tumor growth and decreased expression of Wnt target genes, indicating that loss of sFRP-1 is a pivotal event in renal cell carcinogenesis. These studies strongly suggested that detection of the hypermethylaion status of Wnt antagonist genes could serve as a useful biomarker for RCC. This would be especially valuable if hypermethylation of these genes was an early event in the development of RCC, as has been indicated for other tumors, and if a reliable, non-invasive method to recognize gene hypermethylation were available. Evaluation of gene methylation status in circulating DNA has been described, giving credence to its potential use as a powerful screening tool for cancer diagnosis. However, the minute quantities of DNA in circulation and limited sensitivity of conventional polymerase chain reaction (PCR) methodology are impediments to the advancement of this diagnostic modality. We will utilize real-time PCR technology to develop an assay with increased sensitivity and specificity to detect hypermethylated Wnt antagonist genes in serum samples from patients with RCC. This project will take great advantage of access to RCC samples and expertise from collaborators both inside and outside of the NCI community. Development of a successful protocol to detect Wnt antagonist gene hypermethylation could enable early diagnosis and monitoring of RCC, and perhaps also provide guidance in the choice of cancer therapy, all of which would have profound beneficial effects on patient survival. During the past year various technical issues have raised concern about the reliability of the PCR analysis used for detection of hypermethylated Wnt antagonist genes. We validated the detection of methylated DNA corresponding to the genes encoding sFRP-1, sFRP-4, sFRP-5 and Dkk3 using a combination of nested and real-time PCR. However, we have observed a significant amount of irrelevant PCR product that may be interpreted as a false positive signal. By including a melting curve analysis of PCR product, the likelihood of false positive assignments has been reduced. Based on work with methylated sFRP-1 cDNA, it appears that better discrimination between real and false positive signals may be achieved by omitting the nested PCR step and relying on the inherent differences in melting of PCR products generated from bisulfite treated methylated vs. unmethylated DNA (Lorente et al. BMC Cancer 2008).