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Altered DNA methylation patterns are found in many diseases, particularly in cancer, where the analysis of DNA methylation holds the promise to provide diagnostic, prognostic and predictive information of great clinical value. Methylation of the promoter-associated CpG island of GSTP1 occurs in many hormone-sensitive cancers, has been shown to be a biomarker for the early detection of cancerous lesions and has been associated with important clinical parameters, such as survival and response to treatment. In the current manuscript, we assessed the performance of several widely-used sodium bisulfite conversion-dependent methods (methylation-specific PCR, MethyLight, pyrosequencing and MALDI mass-spectrometry) for the analysis of DNA methylation patterns in the GSTP1 promoter. We observed large discordances between the results obtained by the different technologies. Cloning and sequencing of the investigated region resolved single-molecule DNA methylation patterns and identified heterogeneous DNA methylation patterns as the underlying cause of the differences. Heterogeneous DNA methylation patterns in the GSTP1 promoter constitute a major obstacle to the implementation of DNA methylation-based analysis of GSTP1 and might explain some of the contradictory findings in the analysis of the significance of GSTP1 promoter methylation in breast cancer.

Supporting Information Figures S6 and S7 were incorrectly switched. The image currently appearing as Figure S6 corresponds to the title and legend for Figure S7, and the image currently appearing as Figure S7 corresponds to the title and legend for Figure S6. The titles and legends themselves are in correct order.

IntroductionFew studies have performed expression profiling of both miRNA and mRNA from the same primary breast carcinomas. In this study we present and analyze data derived from expression profiling of 799 miRNAs in 101 primary human breast tumors, along with genome-wide mRNA profiles and extensive clinical information.MethodsWe investigate the relationship between these molecular components, in terms of their correlation with each other and with clinical characteristics. We use a systems biology approach to examine the correlative relationship between miRNA and mRNAs using statistical enrichment methods.ResultsWe identify statistical significant differential expression of miRNAs between molecular intrinsic subtypes, and between samples with different levels of proliferation. Specifically, we point to miRNAs significantly associated with TP53 and ER status. We also show that several cellular processes, such as proliferation, cell adhesion and immune response, are strongly associated with certain miRNAs. We validate the role of miRNAs in regulating proliferation using high-throughput lysate-microarrays on cell lines and point to potential drivers of this process.ConclusionThis study provides a comprehensive dataset as well as methods and system-level results that jointly form a basis for further work on understanding the role of miRNA in primary breast cancer.

Epigenetic changes can be defined as stable molecular alterations of a cellular phenotype such as the gene expression profile of a cell that are heritable during somatic cell divisions (and sometimes germ line transmissions) but do not involve changes of the DNA sequence itself. Epigenetic phenomena are mediated by several molecular mechanisms comprising histone modifications, polycomb/trithorax protein complexes, small non-coding or antisense RNAs and DNA methylation. These different modifications are closely interconnected. Epigenetic regulation is critical in normal growth and development and closely conditions the transcriptional potential of genes. Epigenetic mechanisms convey genomic adaption to an environment thereby ultimately contributing towards given phenotype. In this review we will describe the various aspects of epigenetics and in particular DNA methylation in breast carcinogenesis and their potential application for diagnosis, prognosis and treatment decision.

Glutathione-S-transferase P1 (GSTP1) is involved in thiol-mediated detoxification and breakdown of reactive oxygen species created by anticancer drug exposure. GSTP1 is also an inhibitor of c-Jun N-terminal kinase 1, a kinase involved in stress response, apoptosis and cellular proliferation. Hypermethylation of the GSTP1 promoter has been associated with gene silencing in prostate cancer, kidney cancer, and breast cancer, among others. Although frequently described, the mechanism underlying promoter hypermethylation of the GSTP1 gene is poorly understood. It has been reported that an ATAAA repeat of the GSTP1 promoter separates methylated from unmethylated CpGs in normal prostate tissue [1]. These separate methylation domains are lost in prostate cancer, and methylation extends throughout the whole promoter region. It has been proposed that hypermethylation of GSTP1 requires a combination of gene silencing and random seeds of methylation in prostate cancer cells, and that these combinatorial effects lead to histone deacetylation and subsequent chromatin remodeling [2]. To further elucidate the mechanisms underlying the hypermethylation of the GSTP1 promoter, we genotyped the (ATAAA) repeat and the linked SNPs in positions -354, -288, -287 and -282 in the GSTP1 promoter and we performed methylation analysis using mass spectrometry in tumor DNA from 82 breast cancer patients. The role of the different allelic variants on methylation status of the GSTP1 promoter and expression levels was assessed. We quantitatively determined the methylation status of six CpGs spanning the transcription start site of the GSTP1 promoter: -22, +8, +14, +38, +47 and +55. The average percentage methylation for each individual CpG for the 82 tumor samples analyzed was 16.9%, 30.3%, 18.2%, 21.2%, 18.6% and 8.1%, respectively. The average percentage methylation for all CpGs in all tumor samples was 19%. There was a correlation between the degree of methylation of the individual CpGs and their neighboring CpGs (P < 0.001). When correlating the extent of methylation to the mRNA levels previously assessed by whole genome gene-expression profiling of the same tumors, a significant inverse correlation was observed (P < 0.01). The methylation status of the three CpGs closest to the transcriptional start site was more highly associated with the level of GSTP1 mRNA expression than the CpGs further downstream of the +1 site. Furthermore, we observed differences in the degree of GSTP1 promoter methylation between the different tumor subclasses defined by whole-genome microarray analysis [3]. The methylation of the GSTP1 promoter was significantly lower in the basal subtype compared with the luminal subtype, which corresponded to elevated GSTP1 mRNA levels in the basal subtypes [4]. We further analyzed the impact of the most frequent haplotype structure of the GSTP1 promoter in relation to the extent of methylation, and a correlation was observed (P = 0.003) suggesting that haplotype structures can affect de novo methylation of adjacent sequences.