Epigenetics in Breast Cancer

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Yi Huang; Shweta Nayak; Rachel Jankowitz; Nancy E Davidson; Steffi Oesterreich

Disclosures

Breast Cancer Res. 2011;13(6):225 

In This Article

The DNA Epigenome in Breast Cancer

Single-marker Studies in Breast Cancer

Over the past decade, significant progress has been made in the identification and characterization of altered DNA methylation in breast cancer development and progression. A number of genes have been consistently reported to be methylated, including RASSF1A, ERα, PR, RARβ, CCND2, and PITX2. We will not review these findings here, but would like to point the interested reader towards a number of comprehensive reviews on this topic.[20,58,59] The other frequently hypermethylated gene with a tumor-specific methylation profile is BRCA1.[60,61] Although it has become clear that inactivation of BRCA1 by epigenetic means is a critical event in breast (and ovarian) tumorigenesis, differences in experimental approaches and also in the region of the BRCA1 promoter analyzed resulted in ranges of methylation, and thus warrant some further analysis. In any case, in the present review we will focus on results from some recent genome-wide methylation studies in breast cancer.

Analysis of the Breast Cancer Epigenome Using Genome-wide Approaches

Unprecedented advances have been made in the development of techniques to study genome-wide DNA methylation. Briefly, there are currently four major approaches to identify 5-methylcytosine: restriction endonucleasebased analysis, bisulfite-conversion of DNA, affinity and immunoprecipitation-based studies (methyl Cp6 binding domain (MBD) pulldown, or antibodies against 5-methylcytosine in DNA or against proteins binding to 5-methylcytosine, such as MBD2 and MeCP2), and finally mass spectrometry-based analysis. Most of the approaches have been adapted to be used for genomewide studies using array-based or sequencing-based methods, and some have been utilized to study methylation of the breast cancer genome, as discussed below.

In 2007 Ordway and colleagues used cytosine methylation-dependent restriction enzyme McrBC coupled with array hybridization to analyze methylation in nine matched invasive ductal carcinoma and adjacent normal tissue.[62] They identified 220 differentially methylated loci, and analyzed 16 genes that were able to differentiate breast tumor from normal and benign tissues and blood in more detail. One of these genes was GHSR, a member of the G-protein-coupled receptor that binds to ghrelin. Methylation of GHSR was able to differentiate invasive ductal carcinoma from normal or benign breast tissue with high specificity and sensitivity. The same group went on to study four of the highly methylated loci - GHSR, max gene-associated, nuclear factor I/X, and an unannotated region on chromosome 7 - in more detail through bisulfite pyrosequencing using DNA from breast tumors, normal breast, and sera from cancer patients and from normal controls. Disappointingly, no tumor-specific methylation pattern could be identified, and high methylation rates were detected in normal sera.[63] The latter would clearly pose a problem for the development of sera-based assays and highlights the need to identify markers that are not methylated in normal serum.

Ruike and colleagues reported results from a genome-wide methylated DNA immunoprecipitation sequencing study in breast cancer cells.[64] Briefly, they identified methylated DNA in eight breast cancer cell lines and one normal breast cell line, and in addition they compared methylation rates between parental MCF-7 cells and MCF-7 cells that had undergone epithelial to mesenchymal transition. As expected, the cancer cell lines were characterized by global hypomethylation, concurrent with hypermethylation of many loci. The hypomethylation, which was distributed throughout the entire genome, was three to five times more frequent than hypermethylation, which was clustered at specific loci. Intriguingly, 53% of methylated CpG was found outside CpG islands. Of interest was also the association of epithelial to mesenchymal transition with hypomethylation at many CpG islands, a finding that deserves follow-up.

It will be of great value to apply these approaches to answer clinical questions, such as the association of genome-wide changes in DNA methylation with different grades or stages of breast tumor. A recent genome-wide study by Fang and colleagues[65] has suggested that a CpG island methylator phenotype (CIMP) exists in breast cancer. This breast cancer CIMP provided a distinct epigenomic profile, which was associated with genes that make up the metastasis transcriptome. Additional studies need to be performed before one can confidently state that there are CIMP tumors associated with specific tumor phenotypes. Another interesting question is the association between methylation and molecular subtypes of breast tumors.

A recent study by Holm and colleagues suggests that luminal tumors have higher frequencies of methylation compared with basal or triple-negative breast tumors.[58] Briefly, the authors studied 189 frozen primary breast tumors using the Illumina Golden Gate Methylation Cancer Panel, covering 1,505 CpG loci in 807 cancer-related genes. Unsupervised clustering revealed that methylation patterns were associated with luminal A, luminal B, and basal-like tumors, with luminal B tumors being most methylated and basal-like tumors being the least methylated. As previously reported, Her-2 tumors are very heterogeneous and are mainly driven by amplification of Her-2 as the common denominator. High expression of PRC2 and low methylation of known PRC2 targets in basal-like tumors suggest that PRC2 targets might be silenced by trimethylation of H3K27 in this tumor subset. In general, these data clearly suggest that methylation plays a significant role in the different breast tumor subsets, and it will be critical to determine the mechanism that drives different methylation states. The authors speculate a role for genetic changes in methylation enzymes, an interesting hypothesis that is testable.

Lineage-specific methylation was also observed in a methylation study performed by Sproul and colleagues,[66] who used 27K Infinium arrays to study the methylation at > 14,000 genes in 19 breast cancer cell lines and 47 primary tumors. The authors bring forward the argument that DNA methylation in breast tumors is a marker of cell lineage rather than tumor progression. This study again emphasizes the need to identify tumor-specific methylation events, a task most critical for the future use of DNA methylation for diagnosis and treatment of breast cancer.

Another clinical question is the involvement of DNA methylation in the adaptation of cancer cells to treatment exposure. We recently performed an MBD-pulldown assay in breast cancer cells deprived of estrogen, thus mimicking treatment with aromatase inhibitors. This approach resulted in the identification of a large number of hypermethylated genes, and fewer that were hypomethylated (Pathiraja and colleagues, manuscript in preparation). It will be of great interest to expand those studies to clinical samples, in order to identify markers of resistance, and potential drug targets.

Clearly, these studies are only the beginning for the use of genome-wide methylation studies. With the advent of improved technologies, we should expect to witness an explosion of studies aimed at understanding epigenetic changes in breast cancer. This not only refers to DNA methylation, but also to other epigenetic changes, such as histone modification, which can now also be studied genome wide through the use of chromatin immunoprecipitation technologies At least in part, these efforts should soon benefit from the results of the Human Epigenome Project and TCGA, as briefly discussed below.

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