Ai Lingbao, Tao Qian, Zhong Sheng, Fields C Robert, Kim Wan-Ju, Lee Michael W, Cui Yan, Brown Kevin D, Robertson Keith D
Department of Biochemistry and Molecular Biology and UF-Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Carcinogenesis. 2006 Jul;27(7):1341-8. doi: 10.1093/carcin/bgi379. Epub 2006 Feb 25.
The Wnt signaling pathway is a powerful and prominent oncogenic mechanism dysregulated in numerous cancer types. While evidence from transgenic mouse models and studies of human tumors clearly indicate that this pathway is of likely importance in human breast cancer, few clues as to the exact molecular nature of Wnt dysregulation have been uncovered in this tumor type. Here, we show that the Wnt inhibitory factor-1 (WIF1) gene, which encodes a secreted protein antagonistic to Wnt-dependent signaling, is targeted for epigenetic silencing in human breast cancer. We show that cultured human breast tumor cell lines display absent or low levels of WIF1 expression that are increased when cells are cultured with the DNA demethylating agent 5-aza-2'-deoxycytidine. Furthermore, the WIF1 promoter is aberrantly hypermethylated in these cells as judged by both methylation-specific PCR and bisulfite genomic sequencing. Using a panel of patient-matched breast tumors and normal breast tissue, we show that WIF1 expression is commonly diminished in breast tumors when compared with normal tissue and that this correlates with WIF1 promoter hypermethylation. Analysis of a panel of 24 primary breast tumors determined that the WIF1 promoter is aberrantly methylated in 67% of these tumors, indicating that epigenetic silencing of this gene is a frequent event in human breast cancer. Using an isogenic panel of cell lines proficient or deficient in the DNA methyltransferases (DNMTs) DNMT1 and/or DNMT3B, we show that hypermethylation of the WIF1 promoter is attributable to the cooperative activity of both DNMT1 and DNMT3B. Our findings establish the WIF1 gene as a target for epigenetic silencing in breast cancer and provide a mechanistic link between the dysregulation of Wnt signaling and breast tumorigenesis.
Wnt信号通路是一种强大且显著的致癌机制，在多种癌症类型中均存在失调。虽然来自转基因小鼠模型和人类肿瘤研究的证据清楚地表明，该通路在人类乳腺癌中可能具有重要意义，但在这种肿瘤类型中，关于Wnt失调的确切分子本质的线索却鲜有发现。在此，我们表明，编码一种对Wnt依赖性信号具有拮抗作用的分泌蛋白的Wnt抑制因子-1（WIF1）基因，在人类乳腺癌中成为表观遗传沉默的靶点。我们发现，培养的人类乳腺肿瘤细胞系显示出WIF1表达缺失或水平较低，而当细胞与DNA去甲基化剂5-氮杂-2'-脱氧胞苷一起培养时，WIF1表达会增加。此外，通过甲基化特异性PCR和亚硫酸氢盐基因组测序判断，这些细胞中的WIF1启动子异常高度甲基化。使用一组患者匹配的乳腺肿瘤和正常乳腺组织，我们发现与正常组织相比，乳腺肿瘤中WIF1表达通常会降低，且这与WIF1启动子高甲基化相关。对一组24个原发性乳腺肿瘤的分析确定，这些肿瘤中有67%的WIF1启动子异常甲基化，表明该基因的表观遗传沉默在人类乳腺癌中是一个常见事件。使用一组在DNA甲基转移酶（DNMTs）DNMT1和/或DNMT3B方面 proficient或deficient的同基因细胞系，我们表明WIF1启动子的高甲基化归因于DNMT1和DNMT3B的协同活性。我们的研究结果将WIF1基因确立为乳腺癌中表观遗传沉默的靶点，并在Wnt信号通路失调与乳腺肿瘤发生之间提供了一种机制联系。
