A long-range interactive DNA methylation marker panel for the promoters of HOXA9 and HOXA10 predicts survival in breast cancer patients

Research Open Access A long-range interactive DNA methylation marker panel for the promoters of HOXA9 and HOXA10 predicts survival in breast cancer patients Seong-Min Park†1, 2, Eun-Young Choi†1, Mingyun Bae3, Jung Kyoon Choi3 and Youn-Jae Kim1Email author †Contributed equally Clinical EpigeneticsThe official journal of the Clinical Epigenetics Society20179:73 https://doi.org/10.1186/s13148-017-0373-z ©  The Author(s). 2017 Received: 12 April 2017Accepted: 17 July 2017Published: 24 July 2017 Abstract Background Most DNA cancer methylation markers are based on the transcriptional regulation of the promoter-gene relationship. Recently, the importance of long-range interactions between distal CpGs and target genes has been revealed. Here, we attempted to identify methylation markers for breast cancer that interact with distant genes. Results We performed integrated analysis using chromatin interactome data, methylome data, transcriptome data, and clinical information for breast cancer from public databases. Using the chromatin interactome and methylome data, we defined CpG-distant target gene relationships. After determining the differences in methylation between tumor and paired normal samples, the survival association, and the correlation between CpG methylation and distant target gene expression, we selected CpG methylation marker candidates. Using Cox proportional hazards models, we combined the selected markers and evaluated the prognostic model. We identified six methylation markers in HOXA9 and HOXA10 promoter regions and their long-range target genes. We experimentally validated the chromatin interactions, methylation status, and transcriptional regulation. A prognostic model showed that the combination of six methylation markers was highly associated with poor survival in independent datasets. According to our multivariate analysis, the prognostic model showed significantly better prognostic ability than other histological and molecular markers. Conclusions The combination of long-range interacting HOXA9 and HOXA10 promoter CpGs predicted the survival of breast cancer patients, providing a comprehensive and novel approach for discovering new methylation markers. Keywords BiomarkerPrognosisDNA methylationSurvivalLong-range interactionChromatin interactionHOXA9HOXA10 Background Breast cancer is both the most common cancer and the most frequent cause of cancer-related deaths among women [1]. Based on the expression level of hormone receptors, such as the estrogen receptor (ER) and progesterone receptor (PR), or human epidermal growth factor receptor (Her2), breast cancers are divided into several subtypes, and small molecules or antibodies targeting ER, PR, and Her2 have been used in breast cancer therapies [2]. Breast cancer is conventionally diagnosed by mammography, but this method cannot be applied to some cases, including women with premenopausal breast cancer [3]. Molecular markers and reference laboratory tests for breast cancer diagnosis and prognosis have been developed, but the methods are limited to specific subtypes, such as node-negative and ER-positive breast cancer [4, 5]. Thus, novel approaches for the diagnosis and prognosis of breast cancer are still needed. DNA methylation is one of the most well-known aberrations in human cancers [6]. During tumor progression from normal tissue to invasive cancer, the total level of DNA methylation gradually decreases, but the frequency of hypermethylated CpG islands on promoters increases, causing the transcriptional silencing of tumor-suppressive genes [7, 8]. DNA methylation markers have advantages compared to other molecular markers. For example, hypermethylation of promoter CpGs is a common and early event during the progression of various tumors [9, 10], and DNA methylation is more chemically and biologically stable than RNA or most proteins [6]. DNA methylation markers for cancer diagnosis and prognosis have been discovered, and some of them have been used in clinical trials [11, 12]. For breast cancer, researchers have also reported particular DNA methylation markers [13, 14], some of which need further development for clinical application. The DNA methylation of promoters and CpG islands is known to inhibit target gene expression by regulating the binding of transcription modulators to the promoter [15, 16]. The long-range interaction between CpGs and target genes has been reported [17, 18]. A recent genomic study revealed that the correlation between DNA methylation at distal regulatory sites and long-range target gene expression is significantly stronger than the correlation with promoter methylation and that differences in DNA methylation between cancer and normal tissues at distal regulatory sites are significantly greater than differences in promoter methylation among various cancer types [19]. Nevertheless, most DNA methylation markers for cancer diagnosis and prognosis have been developed based on promoter-gene relationships because of the difficulty of defining the relationship between distal CpGs and target genes. The long-range action of distal CpG-target gene interaction and transcriptional regulation can be specified by chromatin interactome data, particularly data from RNA polymerase II (Pol II) chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) [20]. Thus, novel approaches could be based on the long-range interaction between CpGs and their target genes. In this study, we identified DNA methylation markers for breast cancer and the putative target genes that had long-range interactions using an integrated analysis incorporating the chromatin interactome, methylome, and transcriptome data for breast cancer from public databases. We tried to validate the chromatin interaction, methylation status, and transcriptional regulation. Selected marker candidates were combined to establish a prognostic model and evaluated as markers for breast cancer. Methods Public data analysis The Cancer Genome Atlas (TCGA) methylome (Illumina Infinium Human Methylation 450k BeadChip microarray data, Infinium HM450k) and transcriptome (high-throughput RNA sequencing, RNA-seq) data containing clinical information were downloaded from the International Cancer Genome Consortium (ICGC) data portal (http://icgc.org/). The chromatin interactome (chromatin interaction analysis by paired-end tag sequencing, ChIA-PET) data were downloaded from the Encyclopedia of DNA Elements (ENCODE) databases (https://genome.ucsc.edu/ENCODE/). Another Infinium HM450k methylome dataset for validation was downloaded from the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE39004). The expression microarray (Affymetrix Human Genome U133 Plus 2.0 microarray, affyU133P2) data for MCF7 breast cancer cells after 5-azacytidine (5-aza C) treatment and the untreated control data were downloaded from the NCBI GEO database (GSE22250). The methylome data were globally normali (...truncated)