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.

Portraying breast cancers with long noncoding RNAs

“…By RNA-seq in breast cancer cell lines, two recent studies identified lncRNAs regulated by the ER in the presence or absence of an estrogen agonist ( 43 , 44 ). However, we found only two lncRNAs (DSCAM-AS1 and RP11-161M6.2) that overlap with our ER-related signature and also between these two studies themselves, suggesting differential mechanism between cell lines and tissues and/or specificities related to the method/platform used.…”

Primate-specific oestrogen-responsive long non-coding RNAs regulate proliferation and viability of human breast cancer cells

“…AK057709 has been previously reported as an oestrogen-regulated lncRNA [17], but was not functionally pursued by the team that had first reported it. Oestrogen is a proliferative hormone and hence oestrogen-induced genes are expected to increase cell viability and proliferation.…”

“…The other two leads from Jonsson et al . [17] were LINC01016 (not in our set of 127) and LINC00160, which lacked full-length cDNA support in GenBank at the time of our study design and thus was not represented on our custom microarray. To more globally assess the relationship between our E2-responsive lncRNAs and those from recent studies of ncRNA transcriptome oestrogen responses, we performed a genomic-coordinates, strand-specific, exon-specific intersection of our 127 MCF-7 E2-responsive DE lncRNAs with the much larger catalogue of 1623 lncRNAs identified in human breast cancer subtypes as influencing overall patient survival [18].…”

Biochemical Methods To Investigate lncRNA and the Influence of lncRNA:Protein Complexes on Chromatin

“…Recently, Williams and co-workers used this technique to identify novel estrogen-regulated lncRNAs in breast cancer. 88 …”