title: Single-Molecule Sequencing Reveals Estrogen-Regulated Clinically Relevant lncRNAs in Breast Cancer.
abstract: Estrogen receptor (ER)α-positive tumors are commonly treated with ERα antagonists or inhibitors of estrogen synthesis, but most tumors develop resistance, and we need to better understand the pathways that underlie the proliferative and tumorigenic role of this estrogen-activated transcription factor. We here present the first single-molecule sequencing of the estradiol-induced ERα transcriptome in the luminal A-type human breast cancer cell lines MCF7 and T47D. Sequencing libraries were prepared from the polyadenylated RNA fraction after 8 hours of estrogen or vehicle treatment. Single-molecule sequencing was carried out in biological and technical replicates and differentially expressed genes were defined and analyzed for enriched processes. Correlation analysis with clinical expression and survival were performed, and follow-up experiments carried out using time series, chromatin immunoprecipitation and quantitative real-time PCR. We uncovered that ERα in addition to regulating approximately 2000 protein-coding genes, also regulated up to 1000 long noncoding RNAs (lncRNAs). Most of these were up-regulated, and 178 lncRNAs were regulated in both cell lines. We demonstrate that Long Intergenic Non-protein Coding RNA 1016 (LINC01016) and LINC00160 are direct transcriptional targets of ERα, correlate with ERα expression in clinical samples, and show prognostic significance in relation to breast cancer survival. We show that silencing of LINC00160 results in reduced proliferation, demonstrating that lncRNA expression have functional consequences. Our findings suggest that ERα regulation of lncRNAs is clinically relevant and that their functions and potential use as biomarkers for endocrine response are important to explore.
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Philip
authors.initials: P
authors.lastname: Jonsson
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Cristian
authors.initials: C
authors.lastname: Coarfa
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Fahmi
authors.initials: F
authors.lastname: Mesmar
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Tal
authors.initials: T
authors.lastname: Raz
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Kimal
authors.initials: K
authors.lastname: Rajapakshe
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: John F
authors.initials: JF
authors.lastname: Thompson
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Preethi H
authors.initials: PH
authors.lastname: Gunaratne
authors.affiliation: Center for Nuclear Receptors and Cell Signaling (P.J., F.M., C.W.), Department of Biology and Biochemistry, and Department of Biology and Biochemistry (P.H.G.), University of Houston, Houston, Texas 77204; Molecular and Human Genetics (C.C., K.R.) and Human Genome Sequencing Center (P.H.G.), Baylor College of Medicine, Houston, Texas 77030; Helicos Biosciences (T.R., J.F.T.), Cambridge, Massachusetts 02139; SciLifeLab, School of Biotechnology (C.W.), The Royal Institute of Technology-KTH, 17121 Solna, Sweden; and Department of Biosciences and Nutrition (C.W.), Novum, Karolinska Institutet, 14183 Stockholm, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Williams
mesh_terms: D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000235:genetics*; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020868:Gene Silencing; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D061986:MCF-7 Cells; D062085:RNA, Long Noncoding / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D004967:Estrogens; D062085:RNA, Long Noncoding; D004958:Estradiol