Project description:DNA methylation alterations have similar patterns in normal aging tissue and in cancer. In this study, we investigated breast tissue-specific age-related DNA methylation alterations and used those methylation sites to identify individuals with outlier phenotypes. Outlier phenotype is identified by unsupervised anomaly detection algorithms and is defined by individuals who have normal tissue age-dependent DNA methylation levels that vary dramatically from the population mean. To identify age-dependent DNA methylation sites, we generated DNA methylation sequencing data for 29 purified normal adjacent human breast epithelia (age range 33-82 years old) using Digital Restriction Enzyme Analysis of Methylation (DREAM). Next, we validated the age-related sites in publicly available DNA methylation (450K array) of 97 normal adjacent TCGA samples. We found that hypermethylation in normal breast tissue is the best predictor of hypermethylation in cancer. Using unsupervised anomaly detection approaches, we found that about 10% of the individuals (39 /427) were outliers for DNA methylation from 6 publicly available DNA methylation datasets (GSE88883, GSE74214, GSE101961, GSE69914(normal), GSE69914(normal-adjacent), TCGA (Firehose Legacy)). We also found that there were significantly more outlier samples in normal-adjacent to cancer (24/139, 17.3%) then in normal samples (15/228, 5.2%). Additionally, we found significant differences between predicted ages based on DNA methylation and the chronological ages among outliers and not-outliers. Additionally, we found that accelerated outliers (older predicted age) were more frequent in normal-adjacent to cancer (14/17, 82%) compared to normal samples from individuals without cancer (3/17, 18%). Furthermore, in matched samples, the epigenome of the outliers in the pre-malignant tissue was as severely altered as in cancer.
Project description:Purpose: to characterize epigenetic changes following Twist1 mediated Epithelial-Mesenchymal Transition in human Methods: we characterized the epigenetic and transcriptome landscapes using whole genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results: EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost one-half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRA and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the PRC2 complex, results in blocking EMT and stemness properties. Conclusion: Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb/Trithorax complexes leading to increased cellular plasticity which suggests that its inhibition will prevent EMT, and the associated breast cancer metastasis. DREAM profiles of human mammary epithelial cells before (HMLE_parental) and after Twist1 transfection (HMLE_Twist) were generated in monolayer (HMLE_Twist2D) and sphere culture by deep sequencing using Illumina GAIIx or Illumina hiseq2000. Furthermore, DREAM profile was also obtained in parental human mammary epithelial cells transfected with GFP
Project description:Global proteomic profiling of three mammary epithelial cell types in normal human breast tissue. Primary breast specimens were obtained from 10 women undergoing reduction mammoplasties. Clinical co-variates include age (28-67), hormone status (follicular, luteal, post-menopausal) and mammary epithelial cell type (basal, luminal progenitor, mature luminal).
Project description:Therapeutic targeting MTDH-SND1 interaction Suppresses Breast Cancer Progression and Metastasis (GSE159764). To further elucidate the tumor intrinsic effects of the targeting and the adverse effects on normal mammary epithelial cells Mouse mammary tumor cells and normal mammary epithelial cells were isolated from transgenic mouse models and cultured in vitro 3D spheroids system. MMTV-PyMT tumor cells from cKO mouse in tumorsphere culture were treaed with tamoxifen to induce Mtdh KO, and compared to the mock treatment which maintain wild type Mtdh. Thus the tumor intrinsic effects can be determined Normal mammary epithelial cells were isolated from mouse mammary gland and cultured in vitro in mammosphere. The cells were treated with inhibitor C26A6 vs control and thus the adverse effects on normal MECs can be determined.
Project description:The aim of this project is to examine age-dependent changes in the proteomes and phosphoproteomes of normal human mammary luminal epithelial and myoepithelial cells.
Project description:MDAMB231 cells express the SV40TAg signature. The normal mammary epithelial tissue does not express the signature. CK0082, Empty1 and Gata3 samples are not reported in this manuscript