Wnt Antagonist Gene Hypermethylation in Circulating DNA: Cancer Biomarker

Wnt通路的异常激活是许多类型人类恶性肿瘤的共同特征。一些研究已经证明，在肿瘤细胞中，编码分泌的Wnt拮抗剂的基因经常因高甲基化而沉默，这与这些蛋白质通常作为肿瘤抑制因子的想法一致。肾细胞癌(RCC)中分泌的Frizzled相关蛋白(SFRP)、Dickkopf(DKK)和Wnt抑制因子(WIF)家族的成员被甲基化抑制。此外，肾癌细胞中SFRP-1表达的恢复明显抑制了细胞增殖、肿瘤生长和Wnt靶基因的表达，表明SFRP-1的缺失是肾细胞癌变过程中的关键事件。这些研究有力地表明，检测Wnt拮抗剂基因的超甲基化状态可以作为肾癌的有用生物标志物。如果这些基因的超甲基化是肾癌发展的早期事件，就像其他肿瘤已经指出的那样，并且如果有可靠的、非侵入性的方法来识别基因超甲基化，这将是特别有价值的。已描述了循环DNA中基因甲基化状态的评估，证明其作为一种强大的癌症诊断筛查工具的潜力。然而，循环中微量的DNA和传统聚合酶链式反应(PCR)方法的有限敏感性是这一诊断模式发展的障碍。我们将利用实时荧光聚合酶链式反应技术开发一种灵敏度和特异度更高的检测方法，以检测肾癌患者血清样本中高甲基化的Wnt拮抗剂基因。这个项目将极大地利用从NCI社区内外的合作者那里获得RCC样本和专业知识的机会。开发一种成功的检测Wnt拮抗剂基因高甲基化的方法可以使肾癌的早期诊断和监测成为可能，并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利影响。

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.