Wnt Antagonist Gene Hypermethylation in Circulating DNA: Cancer Biomarker

循环 DNA 中 Wnt 拮抗基因高甲基化：癌症生物标志物

• 批准号: 7965993
• 负责人: Jeffrey Rubin
• 金额: $ 7.58万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965993
• 关键词: Biological Assay; Biological Markers; Blood Circulation; Cell Proliferation; Communities; Complementary DNA; DNA; Detection; Development; Diagnosis; Diagnostic; Discrimination; Early Diagnosis; Evaluation; Event; Family; Gene Targeting; Genes; Human; Hypermethylation; Malignant Epithelial Cell; Malignant Neoplasms; Methodology; Methods; Methylation; Modality; Monitor; Nested PCR; Pathway interactions; Patients; Polymerase Chain Reaction; Proteins; Protocols documentation; Renal Cell Carcinoma; Sampling; Screening procedure; Sensitivity and Specificity; Serum; Signal Transduction; Technology; Time; Tumor Suppressor Proteins; Work; base; bisulfite; cancer diagnosis; cancer therapy; carcinogenesis; frizzled related protein-1; human SFRP4 protein; kidney cell; melting; member; neoplastic cell; prognostic; rat secreted frizzled-related protein 4; response; restoration; tool; tumor; tumor growth

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).

Wnt途径的异常激活是许多类型人类的共同特征 恶性肿瘤。一些研究表明，编码分泌型Wnt的基因 拮抗剂在肿瘤细胞中经常通过超甲基化而沉默，这与 这些蛋白质通常起着肿瘤抑制剂的作用。秘密组织的成员 卷曲相关蛋白（sFRP）、Dickkopf（Dkk）和Wnt抑制因子（WIF）家族是 在肾细胞癌（RCC）中通过高甲基化沉默。此外，sFRP-1的恢复 在RCC细胞中表达显著抑制细胞增殖、肿瘤生长， Wnt靶基因的表达，表明sFRP-1的丢失是肾损伤的关键事件。 细胞致癌作用这些研究强烈表明，检测到超甲基化 Wnt拮抗剂基因的状态可以作为RCC的有用的生物标志物。这将是 如果这些基因的超甲基化是发育的早期事件， RCC的，因为已被指示用于其他肿瘤，如果一个可靠的，非侵入性的方法， 识别基因超甲基化。基因甲基化状态的评估 循环DNA已经被描述，使人们相信它的潜在用途，作为一个强大的 癌症诊断的筛查工具。然而，微量的DNA在循环中， 常规聚合酶链式反应（PCR）方法有限灵敏度是 阻碍了这一诊断模式的发展。我们将利用实时PCR 开发具有增加的灵敏度和特异性的检测技术， 在来自RCC患者的血清样品中检测到高甲基化的Wnt拮抗剂基因。这个项目 将充分利用RCC样本和合作者的专业知识， 在NCI社区内外。开发成功的Wnt检测方案 拮抗剂基因甲基化可使肾癌的早期诊断和监测成为可能， 也许还可以为癌症治疗的选择提供指导，所有这些都将 对患者的生存产生了深远的有益影响。在过去的一年里，各种技术问题 已经引起了对用于检测的PCR分析的可靠性的关注， 高甲基化Wnt拮抗剂基因。我们验证了甲基化DNA的检测 对应于编码sFRP-1、sFRP-4、sFRP-5和Dkk 3的基因， 巢式和实时PCR。然而，我们已经观察到大量不相关的PCR， 可能被解释为假阳性信号的产物。通过加入融化曲线 通过对PCR产物的分析，降低了假阳性分配的可能性。基于 在使用甲基化sFRP-1 cDNA的工作中，似乎可以更好地区分真实的和 假阳性信号可以通过省略巢式PCR步骤并依赖于 由亚硫酸氢盐处理的甲基化产物产生的PCR产物解链的固有差异 与未甲基化DNA相比（Lorente et al. BMC Cancer 2008）。