Supplementary Figures 1-3 from Epithelial Progeny of Estrogen-Exposed Breast Progenitor Cells Display a Cancer-like Methylome

Supplementary Table 1 from Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells

Supplementary Table 2 from Xenoestrogen-Induced Epigenetic Repression of microRNA-9-3 in Breast Epithelial Cells

PDF file - 1049K, Supplementary Figure S1. Summarized sequencing reads and coverage of MBDCap-seq Supplementary Figure S2. Reproducibility test of MBDCap-seq Supplementary Figure S3. Validation of MBDCap-seq by MassARRAY analysis Supplementary Figure S4. Distribution of differentially methylated regions (DMRs) in endometrial tumors relative to normal control Supplementary Figure S5. Network analysis of differential methylation loci by using Steiner-tree analysis Supplementary Figure S6. DNA methylation distribution in early and late stage of the TCGA endometrial cancer cohort Supplementary Figure S7. Relative expression of BMP genes with EGF treatment in the presence or absence of PI3K/AKT and Raf (MAPK) inhibitors in endometrial cancer cells Supplementary Figure S8. Induction of invasion by EGF in AN3CA and HEC1A cell lines Supplementary Figure S9. Knockdown expression of BMP4 and BMP7 in RL95-2 cells Supplementary Figure S10. Relative expression of BMPs and BMPRs in normal endometrial cell and endometrial cancer cell lines Supplementary Figure S11. Microfluidics-based PCR analysis of EMT gene panel in RL95-2 cells with or without EGF treatment Supplementary Figure S12. Knockdown expression of EpCAM by different shRNA sequences in RL95-2 cells Supplementary Figure S13. Proposed model for EpICD mediated regulation of BMP genes by EGF Supplementary Table 1. Characteristics of endometrial cancer patients Supplemental Table 2. Summary of sequencing reads in endometrial tumors an normal controls Supplementary Table 3. Differentially methylated promoter CpG islands in recurrent relative to non-recurrent tumors Supplementary Table 4. List of primer sequences Supplementary Table 5. List of siRNA and shRNA sequences

Supplementary Table 1 from Epithelial Progeny of Estrogen-Exposed Breast Progenitor Cells Display a Cancer-like Methylome

Nuclear radiation and radioactive fallouts resulting from a nuclear weapon detonation or reactor accidents could result in injuries affecting multiple sensitive organs, defined as acute radiation syndrome (ARS). Rapid and early estimation of injuries to sensitive organs using markers of radiation response is critical for identifying individuals who could potentially exhibit ARS; however, there are currently no biodosimetry assays approved for human use. We developed a sensitive microRNA (miRNA)-based blood test for radiation dose reconstruction with ±0.5 Gy resolution at critical dose range. Radiation dose-dependent changes in miR-150-5p in blood were internally normalized by a miRNA, miR-23a-3p, that was nonresponsive to radiation. miR-23a-3p was not highly expressed in blood cells but was abundant in circulation and was released primarily from the lung. Our assay showed the capability for dose estimation within hours to 1 week after exposure using a drop of blood from mice. We tested this biodosimetry assay for estimation of absorbed ionizing radiation dose in mice of varying ages and after exposure to both improvised nuclear device (IND)-spectrum neutrons and gamma rays. Leukemia specimens from patients exposed to fractionated radiation showed depletion of miR-150-5p in blood. We bridged the exposure of these patients to fractionated radiation by comparing responses after fractionated versus single acute exposure in mice. Although validation in nonhuman primates is needed, this proof-of-concept study suggests the potential utility of this assay in radiation disaster management and clinical applications.

Hepatocellular carcinomas (HCC) exhibit distinct promoter hypermethylation patterns, but the epigenetic regulation and function of transcriptional enhancers remain unclear. Here, our affinity- and bisulfite-based whole-genome sequencing analyses reveal global enhancer hypomethylation in human HCCs. Integrative epigenomic characterization further pinpoints a recurrent hypomethylated enhancer of CCAAT/enhancer-binding protein-beta (C/EBPβ) which correlates with C/EBPβ over-expression and poorer prognosis of patients. Demethylation of C/EBPβ enhancer reactivates a self-reinforcing enhancer-target loop via direct transcriptional up-regulation of enhancer RNA. Conversely, deletion of this enhancer via CRISPR/Cas9 reduces C/EBPβ expression and its genome-wide co-occupancy with BRD4 at H3K27ac-marked enhancers and super-enhancers, leading to drastic suppression of driver oncogenes and HCC tumorigenicity. Hepatitis B X protein transgenic mouse model of HCC recapitulates this paradigm, as C/ebpβ enhancer hypomethylation associates with oncogenic activation in early tumorigenesis. These results support a causal link between aberrant enhancer hypomethylation and C/EBPβ over-expression, thereby contributing to hepatocarcinogenesis through global transcriptional reprogramming.

Early postnatal exposures to Bisphenol A (BPA) and genistein (GEN) have been reported to predispose for and against mammary cancer, respectively, in adult rats. Since the changes in cancer susceptibility occurs in the absence of the original chemical exposure, we have investigated the potential of epigenetics to account for these changes. DNA methylation studies reveal that prepubertal BPA exposure alters signaling pathways that contribute to carcinogenesis. Prepubertal exposure to GEN and BPA + GEN revealed pathways involved in maintenance of cellular function, indicating that the presence of GEN either reduces or counters some of the alterations caused by the carcinogenic properties of BPA. We subsequently evaluated the potential of epigenetic changes in the rat mammary tissues to predict survival in breast cancer patients via the Cancer Genomic Atlas (TCGA). We identified 12 genes that showed strong predictive values for long-term survival in estrogen receptor positive patients. Importantly, two genes associated with improved long term survival, HPSE and RPS9, were identified to be hypomethylated in mammary glands of rats exposed prepuberally to GEN or to GEN + BPA respectively, reinforcing the suggested cancer suppressive properties of GEN.

Aberrant DNA methylation at promoters is often linked to tumorigenesis. But many aspects of DNA methylation remain unexplored, including the individual roles of distal and gene body methylation, as well as their collaborative roles with promoter methylation. Here we performed a MBD-seq analysis on prostate specimens classified into low, high, and very high risk group based on Gleason score and TNM stages. We identified gene sets with differential methylation regions (DMRs) in Distal, TSS, gene body and TES. To understand the collaborative roles, TSS was compared with the other three DMRs, resulted in 12 groups of genes with collaborative differential methylation patterns (CDMPs). We found several groups of genes that show opposite methylation patterns in Distal and Genic regions compared to TSS region, and in general they are differentially expressed genes (DEGs) in tumors in TCGA RNA-seq data. IPA (Ingenuity Pathway Analysis) reveals AR/TP53 signaling network to be a major signaling pathway, and survival analysis indicates genes subsets significantly associated with prostate cancer recurrence. Our results suggest that DNA methylation in Distal and Genic regions also plays critical roles in contributing to prostate tumorigenesis, and may act either positively or negatively with TSSs to alter gene regulation in tumors.

Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each other's binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

BACKGROUND:Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR).METHODS:Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses.RESULTS:Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy.CONCLUSIONS:Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. .

Objectives: This retrospective chart review was designed to determine the difference in chemotherapy (CT) treatment and clinical trial enrollment practices by gynecologic oncologists (GYN) and medical oncologists (MED) involved in the treatment of recurrent metastatic (RM) uterine leiomyosarcoma (uLMS) at an institution with a sarcoma center.

Objective. A retrospective chart review was performed to determine patient outcomes before and after partnership by gynecologic oncologists (GYN/ONC) with a sarcoma center (SC) for patients with recurrent unresectable/metastatic (RM) uterine leiomyosarcoma (uLMS).Methods. 58 RM patients, identified from medical records of uLMS patients cared for by either GYN/ONC service and/or the SC between 1/1/2000-4/1/2014, were audited for patient and tumor characteristics, outcomes, and clinical trials enrollments.Results. Of the 58 patients, 26 patients (48%) were treated by GYN/ONC alone and 32 were treated by a combination of GYN/ONC and SC (52%). Age, race, tumor size, grade, presence of lymphovascular invasion, cervical involvement, and FIGO stage at diagnosis were not statistically different between the two groups. There was a significant difference between the number of clinical trial enrollments (0.07 vs 0.84 trials/ patient, p < 0.001) and the number of chemotherapy regimens prescribed (2.67 vs 4.29/patient, p = 0.03) by GYN/ONC vs SC; the latter was driven by the number of clinical trial enrollments by the SC. Sixty-nine percent of patients referred to the SC were enrolled in at least one clinical trial, while just 8% of patients in the GYN/ONC group were enrolled in at least one clinical trial, a difference that is significant (p < 0.0001).Conclusions. Referral of RM uLMS patients by GYN/ONC to a dedicated clinical trials-based SC resulted in an increase in the number of chemotherapy regimens prescribed and clinical trial enrollments. Partnership between GYN/ONC and a dedicated SC with access to clinical trials should be encouraged for all RM uLMS patients. (C) 2015 Elsevier Inc. All rights reserved.

Tau undergoes different types of modifications, some of which have been found to coincide with neurodegenerative pathology. Studies have shown that caspase-mediated truncation of tau at the C-terminal leads to extensive filament formation and NFT development. Here, we examine the neurodegenerative effects of full length (FL-tau) and C-terminal truncated D422-441aa tau (C-tau) in young and old non-transgenic (ntg) mice in an effort to characterize the structural changes associated with tau polymerization and progression to pathology. Ntg animals at ages 4 and 11 month-old received bilateral injections of FL-tau and C-tau delivered via recombinant adeno-associated 9 (AAV9) vector in both hippocampai and cortices. Levels of total tau expression, Tau pathology, synaptic and neuronal integrity were measured in the brain tissues of ntg mice from both age groups using imunohistochemistry (IHC) and Western Blots (WB) analysis. Cognitive changes in experimental and control animals were assessed in radial arm water maze (RAWM). Expression of FL-tau and C-tau increased significantly with age (p<0.05), albeit no significant difference between both Tau isoforms was measured at the 11 month-old group. Interestingly, levels of pSer202/ pThr205 were significantly higher in the cortex and hippocampal regions of 11 month-old C-Tau injected mice compare to the 4 month old group (p<0.01) and to the 11 month-old FL-Tau injected mice (p<0.05). Aged FL-tau injected mice showed moderate levels of phosphorylated epitopes reaching significance in cortex (p<0.05). Significant loss in cortical layer I-VI and hippocampal granular and pyramidal layers was measured by NeuN stain in the old animals expressing both Tau forms (p<0.01). Western Blot analysis showed significant increases in total Tau levels (p<0.05) and pS262 (p<0.05) in 11month-old C-tau expressing mice. Analysis for synaptic changes following FL-Tau and C-Tau expression demonstrated significant reduction of synaptophysin levels in aged animals (p<0.05). RAWM test revealed an age effect between C-Tau expressing animals and the control group (p<0.05). Our data demonstrate that age is a key factor in progression of Tau pathology following expression of FL-Tau and C-Tau isoforms, and correlated with development of neurodegenerative events such as neuronal loss, synaptic deficit. Meanwhile, C-Tau expression in ntg mice promoted cognitive deficits with age.

Recent genome-wide analysis has shown that DNA methylation spans long stretches of chromosome regions consisting of clusters of contiguous CpG islands or gene families. Hypermethylation of various gene clusters has been reported in many types of cancer. In this study, we conducted methyl-binding domain capture (MBDCap) sequencing (MBD-seq) analysis on a breast cancer cohort consisting of 77 patients and 10 normal controls, as well as a panel of 38 breast cancer cell lines.

Introduction: Multiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of plasma cells (PCs) in the bone marrow (BM). Over 50% of patients die within 5 years of diagnosis. The transition from normal PCs to active MM, via premalignant condition (monoclonal gammopathy of undetermined significance; MGUS), consists of many oncogenic events, including upregulation of cyclin D1 and IRF4 genes, as well as activating mutations in NRAS, and KRAS. Despite recent advances in oncogenomics, further studies are needed to identify critical players in MM pathogenesis that could be targeted for pharmacological intervention to improve outcome. Recently, aberrations in epigenetic modifications, including DNA modifications and miRNA expression have been shown to play a crucial role in development and progression of MM. miRNAs are short non-coding RNAs that bind to complementary sequences on target messenger RNA (mRNAs) and downregulate their expression by inhibiting mRNA translation, or inducing its degradation. miRNA analysis, may lead to improved understanding of MM biology and classification, by establishing associations between gene expression changes and MM molecular and clinical features. To assess whether miRNA deregulation plays a critical role in the development of MM we performed small RNA next generation sequencing in the PCs isolated from 3 patients at the MGUS stage and after that they developed active disease, but still untreated. miRNA deregulation was also validated in an independent set of newly diagnosed MM patients (n.34) compared to non-cancer age matched donors (n.6). Mechanisms of transcriptional regulation and biological roles of the differentially expressed miRNAs were also analyzed.

Abstract Recently, we have described that the breast of parous postmenopausal women exhibits a specific gene expression profile induced by full term pregnancy (FTP) [Belitskaya-Levy, I. et al. 2011 and Peri, S. et al. 2012], which is centered on chromatin remodeling [Russo, J. 2012]. In order to better understand the molecular mechanism of gene regulation induced by FTP, we have investigated the DNA methylation profile in breast core needle biopsies of five nulliparous (NP) and five parous (P) postmenopausal women between the ages of 50 and 69. DNA was extracted from core needle biopsies and highly methylated fragments of the DNA were separated from the un-methylated DNA using MBD-Capture. The hypermethylated DNA was then sequenced using the Illumina system. Image analysis and base calling were performed with the standard Illumina pipeline, and the reads were aligned to human reference genome (hg18). Differentially methylated regions (DMR) were identified by comparing the differences of averaged methylation values between P and NP samples using Student's t-test (P<0.01). 583 genes were differently methylated between P and NP, of which, 455 genes were hypermethylated in the P breast and 128 genes were hypermethylated in the NP. Additional visual analyses of the sequencing results were performed in these 583 genes using Integrative Genomics Viewer (IGV), of which, 53 genes with DMRs were identified. Analysis and research into the functions of these genes identified seven genes (DACT1, PPP2CA, GSK3B, ROBO1, INPP4B, IL6ST, FZD1) that have been described to interact with each other in either the Wnt signaling pathway or its controlling PI3K/AKT/mTOR pathways. The evaluation of these interactions suggests that beta-catenin, a downstream gene of the Wnt signaling pathway, is being inhibited in the P women. Wnt signaling plays different roles during the stages of the mammary gland development, and it is suggested that lowered beta-catenin expression is essential for proper mammary involution. This observed molecular machinery, leading to a decrease in beta-catenin production and accumulation, may be a leftover effect from mammary involution, last process that the parous mammary gland undergone and it may be, at least partially, responsible for their decreased risk of cancer. The pathways identified in this specific population are of the great importance and could represent a safeguard mechanism mediating the protection of the breast conferred by full term pregnancy. (The sample collection was supported by Avon Foundation for Women Breast Cancer Research Program grant 02-2010-117 and the methylation studies by NIH core grant CA06927 to Fox Chase Cancer Center). Citation Format: Julia Santucci-Pereira, Colleen O'Malley, Ricardo López de Cicco, Nameer B. Kirma, Tim H. Huang, Joseph Liu, Eric A. Ross, Michael Slifker, Suraj Peri, Irma H. Russo, Pal Bordas, Per Lenner, Göran Hallmans, Paolo Toniolo, Jose Russo. Pregnancy changes the DNA methylation profile of the breast in postmenopausal women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2294. doi:10.1158/1538-7445.AM2014-2294

Abstract Endometrial cancer (EC) is the most common gynecologic malignancy, with approximately 20% of EC patients developing advanced stage recurrent tumors and frequent metastasis. Our goal was to investigate whether DNA methylation signatures associated with low promoter methylation (hypomethylation) and specific oncogenic signaling delineate predictive markers of endometrial cancer recurrence. Global screening by Methyl-CpG-capture sequencing revealed aberrant DNA methylation in our endometrial cancer cohort and identified a subset of bone morphogenetic protein family (BMP1, 2, 3, 4, and 7) exhibiting frequent hypomethylation in primary tumors with subsequent recurrence compared with non-recurrent tumors. This epigenetic signature correlated with poor survival and was validated in The Cancer Genome Atlas endometrial cancer cohort. Our functional studies also implicated epidermal growth factor receptor (EGFR) pathway in the transcriptional activation of these BMPs and inducing epithelial-mesenchymal transition (EMT). In addition to AKT and MAPK, the epithelial cell adhesion molecule (EpCAM) mediated these actions by EGFR. EpCAM involvement in cancer progression includes nuclear co-translocation of its intracellular domain EpICD with Lef-1 complexes and targeting oncogene promoter activation. In this study, EGF stimulated EpICD-Lef-1 binding on BMP genes accompanied with histone active modification marks. EpICD knockdown resulted in increased repressive histone marks and DNA methylation at these loci, suggesting that EpICD occupancy is involved in their epigenetic modification to an open transcriptional conformation. Knockdown of candidate BMPs led to decreased endometrial cancer cell invasiveness, implicating them in aggressive growth. Extending our studies, we performed ChIP-Seq analysis to identify global regulation by the EpICD-Lef-1 complexes in endometrial cancer. Interestingly, under basal levels only about 28% of loci targeted by either EpICD or Lef-1 were commonly targeted by EpICD-Lef-1, with common targets increasing to about 50% in 24 hrs and 73% in 48 hrs post EGF treatment. This indicates that EGFR signaling stimulates a time-course dependent enrichment of EpICD-Lef-1 convergence on target loci. Our initial studies of these EpICD target pathways included genes with cell adhesion functions. Future studies on the regulation of the EpICD-Lef-1 regulated genes and their mechanisms of action in aggressive endometrial cancer will provide a better understanding of this gene network in endometrial cancer. Hypomethylation signatures of candidate loci in this regulatory network may present putative predictive markers of poor survival and which may be used to tailor individualized therapy. Citation Format: Ya-Ting Hsu, Fei Gu, Yi-Wen Huang, Joseph Liu, Jianhua Ruan, Rui-Lan Huang, Chiou-Miin Wang, Chun-Liang Chen, Rohit R. Jadhav, Yao Wang, Victor X. Jin, Hung-Cheng Lai, David G. Mutch, Paul J. Goodfellow, Ian M. Thompson, Nameer B. Kirma, Tim H.M. Huang. EpCAM-mediated hypomethylation of BMP and cell adhesion genes is associated with advanced endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1376. doi:10.1158/1538-7445.AM2014-1376