Luminal lncRNAs Regulation by ERα-Controlled Enhancers in a Ligand-Independent Manner in Breast Cancer Cells

Abstract

Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal BC phenotype. Recently, we demonstrated that even in absence of ligands ERα (apoERα) binds chromatin sites where it regulates transcription of several protein-coding and lncRNA genes. Noteworthy, apoERα-regulated lncRNAs marginally overlap estrogen-induced transcripts, thus representing a new signature of luminal BC genes. By the analysis of H3K27ac enrichment in hormone-deprived MCF-7 cells, we defined a set of Super Enhancers (SEs) occupied by apoERα, including one mapped in proximity of the DSCAM-AS1 lncRNA gene. This represents a paradigm of apoERα activity since its expression is largely unaffected by estrogenic treatment, despite the fact that E2 increases ERα binding on DSCAM-AS1 promoter. We validated the enrichment of apoERα, p300, GATA3, FoxM1 and CTCF at both DSCAM-AS1 TSS and at its associated SE by ChIP-qPCR. Furthermore, by analyzing MCF-7 ChIA-PET data and by 3C assays, we confirmed long range chromatin interaction between the SE and the DSCAM-AS1 TSS. Interestingly, CTCF and p300 binding showed an enrichment in hormone-depleted medium and in the presence of ERα, elucidating the dynamics of the estrogen-independent regulation of DSCAM-AS1 expression. The analysis of this lncRNA provides a paradigm of transcriptional regulation of a luminal specific apoERα regulated lncRNA.

DSCAM-AS1-Driven Proliferation of Breast Cancer Cells Involves Regulation of Alternative Exon Splicing and 3′-End Usage

“…We previously demonstrated that DSCAM-AS1 is an ERα-regulated lncRNA [ 9 , 13 ]. We thus compared the list of DE genes obtained in this study with those DE upon ERα silencing [ 13 ]. Notably, 526 genes were detected as DE in both datasets (hypergeometric test p < 4.36 × 10 −48 , representation factor = 1.7) ( Figure 2 e and Table S4 ).…”

“…It is interesting to note that DSCAM-AS1 was also detected as highly expressed in HER2-amplified, ER-negative tumors, suggesting an ERα-independent mechanism enhancing its expression in this BC subtype. As previously shown by our group, a super-enhancer region is localized upstream of the DSCAM-AS1 locus and it is involved in the regulation of this lncRNA [ 13 ]. This genomic region can represent a platform for the recruitment of different transcriptional regulators, including the Transcription Factor AP2-γ (AP2-γ), which was previously reported to lead overexpression of the ERBB2 gene [ 32 ] and, in complex with other transcription factors, it could drive the DSCAM-AS1 overexpression.…”

“…We chose LNA GapmeRs since, in comparison with siRNAs, they were demonstrated to be more stable, more efficient in targeting nuclear RNAs, and they rely on the activation of RNaseH, which cleaves the RNA:LNA hybrids, avoiding the saturation of silencing machineries, like the siRNA/AGO complex [ 33 ]. Strikingly, by comparing the gene expression changes occurring upon DSCAM-AS1 silencing to those occurring upon ERα silencing [ 34 ], both DSCAM-AS1 and ERα were predicted to be involved in the same pathway regulating cell growth and survival of MCF-7 BC cells, consistent with the role of DSCAM-AS1 as a downstream effector of the ERα signaling pathway [ 13 ]. These results are in line with those from Sun and colleagues, reporting that siRNA-mediated DSCAM-AS1 silencing induces cell cycle arrest in MCF-7 cells [ 11 ].…”

“…The number of genes per GO (gene ontology) term is shown and the color code is proportional to significance. ( e ) Venn Diagram showing the overlap between genes DE in this study and those DE upon ERα silencing [ 13 ]. ( f , g ) Top 20 enriched GO terms of overlapping DE genes showing concordant regulation are shown for genes upregulated ( f ) and downregulated ( g ) in both datasets.…”

“…Niknafs and colleagues reported the overexpression of the lncRNA in tamoxifen-resistant cells whose proliferation decreases upon DSCAM-AS1 silencing [ 10 ]. Alongside these results, our group showed that, in BC cells grown in hormone-deprived medium, ERα binding occurs in a super-enhancer region upstream of the DSCAM-AS1 locus, which promotes the lncRNA overexpression in these cells [ 13 ]. These results, in addition to the evidence of DSCAM-AS1 overexpression in BC patients with poor outcomes [ 11 ], make the understanding of the functional role of this lncRNA relevant.…”