Epigenetics in Breast Cancer

What's New?

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

Disclosures

Breast Cancer Res. 2011;13(6):225 

In This Article

Epigenome Project and TCGA: Their Role in Understanding Epigenetics of Breast Cancer (and Other Diseases)

Description of Epigenome Roadmap Initiatives

The Epigenome Project is a Roadmap initiative led by several National Institutes of Health (NIH) centers, started in 2008 when the NIH decided to invest over $190 million to accelerate the advancement of biomedical research in epigenomics. A series of five initiatives was therefore created. The first initiative is the creation of reference epigenome mapping centers, which support the development of reference epigenomes of a variety of human cells, including normal breast epithelial cells. The data gathered include information on DNA methylation, histone modifications, and associated noncoding RNAs. The second initiative focuses not only on coordinating the banking of data, but also on facilitating its access to the public, accomplished through the creation of the Epigenomic Data Analysis and Coordination center. The third initiative seeks to advance technology in epigenetic research, by enabling the development of new techniques, including the creation of methods that allow in vivo imaging of epigenetic changes. The objective of the fourth initiative is to identify epigenetic marks and establish their function in mammalian cells. Finally, the intention of the fifth initiative is to identify those epigenetic changes that are the cause of specific diseases, including breast cancer.

Importantly, the NIH roadmap initiative is part of an international association, the International Human Epigenome Consortium, which has made several recommendations with regard to data release, format, and various technical considerations, in order to universalize and validate findings.[67] Regarding the former, it is recommended that all data be made available through one of several public databases, such as GEO, ARRAYEXPRESS, and DDBJ.

Funded roadmap initiatives have resulted in many fundamental contributions, including a study published late in 2009 that presented the first genome-wide, singlebase resolution map of methylated cytosines in the mammalian genome from both human embryonic stem cells and fetal fibroblasts.[68] Importantly, almost onequarter of all methylation identified in stem cells was in a non-CpG context, a finding that does not seem to be restricted to methylation in stem cells. Subsequently, an approach was developed to sequence chromatin-immunoprecipitated DNA from limited cell populations, an approach most critical for working with clinical samples.[69] A study by Ernst and Kellis described a multivariate Hidden Markov Model to reveal chromatin states in human T cells through the systematic analysis of 51 chromatin states, including promoter-associated states, transcription-associated states, active intergenic states, large-scale repressed states, and repeat-associated states.[70]

Finally, two studies have explored the strengths and weaknesses of four methods of DNA methylation mapping technologies, while providing recommendations on the design of case-control studies in epigenomics.[71,72] These studies mark a critical milestone for the Human Epigenome Project, since the development of genome-wide technology has been a major focus on the initiative. Briefly, six methods were tested, of which five were sequence-based and one was array-based. Each method was subjected to rigorous testing, and to statistical analysis of at least two replicate samples. Although resolution and coverage differed, there was high concordance between the different methods, providing a high level of confidence for all epigenetic researchers, and providing flexibility as to which methods to choose based on the need for resolution, the amount of available starting material, and, last but not least, the budget.

NIH Roadmap Studies Deciphering the Breast Cancer Epigenome

The initiative also funded disease-specific studies, including those in breast cancer. One such study is the analysis of special AT-rich sequence binding 1 (SATB1) in metastatic breast cancer. The Kohwi-Shigematsu laboratory identified SATB1, originally described as a genome organizer in thymocytes,[73–75] to be a key determinant in breast cancer metastasis.[76]

While there is some controversy about the detailed function of SATB1 in breast cancer,[77] there is no doubt that SATB1 functions as a critical, global, genome organizer by recognizing and binding to specialized DNA sequences in the genome that have a high propensity to unwind (base-unpairing regions). SATB1 organizes chromatin into loops through binding of base-unpairing regions, which are found in gene-rich regions, and can regulate a large number of distally located genes by functioning as a landing platform for multiple chromatin remodeling/modifying proteins that confer specific epigenetic marks.[78] In breast cancer, once SATB1 becomes expressed, it regulates ~1,000 genes, including those involved in cancer progression, metastasis, and growth (for example, ERBB2, transforming growth factor beta).

The Kohwi-Shigematsu group is currently using genome-wide approaches to map all base-unpairing regions in the genome and to determine which particular subset of these is bound by SATB1, and whether these specific epigenomic modifications are associated with poor-prognosis expression profiles of aggressive breast cancers. In addition, using a new approach of analyzing three-dimensional gene interactions, they have found that the c-MYC gene locus is frequently brought into close proximity with a multitude of genes related to myc or co-amplified in cancer. Forced SATB1 expression in nonaggressive breast cancer cells led to a major change in c-MYC interaction pattern, establishing new connections with genes, some of which are related to cancer (T Kohwi-Shigematsu, personal communication). This study provides a concrete example of how the Epigenome Project supports the understanding of the progression from nonaggressive breast cancer to metastatic cancer by establishing genome-wide changes in epigenetic marks, at least in part through global reorganization of higher order chromatin structures by proteins such as SATB1 and others. The critical role for chromatin-organizing proteins is reflected by frequent mutations, such as the recently described mutation of ARID1A in ovarian cancer[47] and other cancers.[79]

In addition, beginning in October 2010, the Epigenome Project began to release data that included more than 300 maps of epigenetic changes in over 56 cell and tissue types, including a number of normal breast cells.[80] For example, one can access data on DNA methylation as well as a number of critical genome-wide histone markers (for example, methylation at lysines K4, K9, K27, and K36). These data should help to define breast cancerspecific changes, by allowing researchers to compare the breast cancer data with the normal reference epigenome. This, however, brings up one of several hurdles the project must overcome. There are challenges regarding data integration, interpretation, and dissemination - as one would expect given that the technologies used are all relatively new. There is a critical need for the creation of a new generation of tools for interpretation of the numerous epigenetic datasets.[81] Briefly, in contrast to DNA sequence data, epigenomic data are not digital, differ in resolution, and are highly variable. These features make comparisons of epigenomes challenging, and require sophisticated informatics tools often not easily accessible for just any general laboratory. Increasing accumulation of data, coupled with improved data and tool integration, and access to computing resources and services, preferably through well-established and proven pipelines, are necessary for the efficient and successful analysis of the unprecedented increase of epigenetic information.

Epigenetic Studies in Breast Cancer as Part of the Cancer Genome Atlas

TCGA began in 2006 as a combined effort by the National Cancer Institute and the National Human Genome Research Institute. The success of the three-year pilot project led the NIH to commit major resources to TCGA to collect and characterize more than 20 tumor types, including breast cancer. Tumor DNA and RNA will be thoroughly characterized using a number of approaches. Data are currently available for the brain tumor glioblastoma multiforme (GBM) and ovarian cancer, and we can expect more completion of the breast cancer studies by the end of this year.

Currently, epigenomics studies within TCGA use the HumanMethylation27 BeadChip (Illumina, San Diego, CA, USA). This assay allows quantitative interrogation of 27,578 CpG loci covering more than 14,000 genes at single-nucleotide resolution. Specifically, the panel targets CpG sites located within the proximal promoter regions of 14,475 consensus coding sequences, and in 110 miRNA promoters. As of April 2011 these data have been available from the TCGA data portal for more than 400 breast cancers,[82] and we can look forward to the report of its first analysis. Of note, a similar TCGA-directed approach in GBM resulted in the identification of a unique glioma CIMP in about 10% of patients, who are usually very young at the time of diagnosis. These patients survive more than 3 years, which is in stark contrast to most GBM patients who survive fewer than 15 months. Interestingly, the study also revealed an association between glioma CIMP with an acquired mutation in the IDH1 gene.

The development of these technologies is moving very rapidly, and just when we thought we had a battery of gold standards for genome-wide analysis of DNA methylation it becomes clear that additional modifications such as hydroxymethyl cytosines, and methylated cytosines outside mCpG islands and outside promoter regions, are likely to play critical roles, including in breast cancer. One may have speculated that the sole analysis of promoter methylation - as done in the TCGA studies - might miss cancer-specific changes in other critical regulatory regions. The exciting findings from the GBM study, however, clearly show that promoter methylation includes clinically significant information, and we should look forward to additional data and analyses from the breast cancer TCGA studies.

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