Rauscher Garth H, Kresovich Jacob K, Poulin Matthew, Yan Liying, Macias Virgilia, Mahmoud Abeer M, Al-Alem Umaima, Kajdacsy-Balla Andre, Wiley Elizabeth L, Tonetti Debra, Ehrlich Melanie
Division of Epidemiology and Biostatistics, University of Illinois-Chicago, School of Public Health, M/C 923, Chicago, IL, 60612, USA.
EpigenDx, Inc., Hopkinton, MA, USA.
BMC Cancer. 2015 Oct 29;15:816. doi: 10.1186/s12885-015-1777-9.
Breast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation.
Pyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites.
Most of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 - 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the gene structure was further illustrated by bioinformatic analysis of an alternative promoter/intron gene region for APC.
We confirmed the frequent DNA methylation changes in invasive breast cancer at a variety of genome locations and found evidence for an extensive field effect in breast cancer. In addition, we illustrate the power of combining publicly available whole-genome databases with a candidate gene approach to study cancer epigenetics.
乳腺癌的形成与DNA甲基化的频繁变化相关，但DNA甲基化非常早期改变的程度以及癌症相关表观遗传变化的生物学意义仍需进一步阐明。
对来自福尔马林固定、石蜡包埋切片的亚硫酸氢盐处理DNA进行焦磷酸测序，这些切片包含浸润性肿瘤以及与癌症相邻的组织学正常组织的配对样本，还有来自未受影响女性的对照性缩乳术样本。所研究的DNA区域包括启动子（BRCA1、CD44、ESR1、GSTM2、GSTP1、MAGEA1、MSI1、NFE2L3、RASSF1A、RUNX3、SIX3和TFF1）、远上游区域（EN1、PAX3、PITX2和SGK1）、内含子（APC、EGFR、LHX2、RFX1和SOX9）以及LINE-1和卫星2 DNA重复序列。这些选择基于先前的文献或公开可用的DNA甲基化组图谱。甲基化百分比是在相邻的CpG位点上平均得出的。
与相邻组织相比，大多数检测的基因区域在癌症中显示出高甲基化，但TFF1和MAGEA1区域显著低甲基化（p≤0.001）。重要的是，在大量患者（105 - 129例）以及15 - 18例对照性缩乳术样本中检测的16个区域中的6个区域，在相邻的组织学正常组织与非癌性缩乳术样本相比时已经异常甲基化（p≤0.01）。此外，对转录组和DNA甲基化数据库的检查表明，三个非启动子区域（远上游的EN1和PITX2以及内含子LHX2）的甲基化与较高的基因表达相关，这与通常报道的癌症DNA高甲基化与癌症改变基因表达之间的负相关不同。这三个非启动子区域在正常组织中也表现出与分化相关基因表达呈正相关的组织特异性高甲基化（在肌肉祖细胞与许多其他类型的正常细胞相比时）。对APC的一个替代启动子/内含子基因区域的生物信息学分析进一步说明了考虑所分析的确切DNA区域和基因结构的重要性。
我们证实浸润性乳腺癌在多种基因组位置存在频繁的DNA甲基化变化，并发现了乳腺癌中广泛的场效应的证据。此外，我们展示了将公开可用的全基因组数据库与候选基因方法相结合来研究癌症表观遗传学的作用。
