A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer.

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein-protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

The Estrogen Receptor α Signaling Pathway Controls Alternative Splicing in the Absence of Ligands in Breast Cancer Cells.

BACKGROUND: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. METHODS: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. RESULTS: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3'end processing. ApoERα depletion results in 758 isoform switching events with effects on 3'end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. CONCLUSION: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level.

RNA modifications detection by comparative Nanopore direct RNA sequencing.

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

The non-coding epitranscriptome in cancer.

Post-synthesis modification of biomolecules is an efficient way of regulating and optimizing their functions. The human epitranscriptome includes a variety of more than 100 modifications known to exist in all RNA subtypes. Modifications of non-coding RNAs are particularly interesting since they can directly affect their structure, stability, interaction and function. Indeed, non-coding RNAs such as tRNA and rRNA are the most modified RNA species in eukaryotic cells. In the last 20 years, new functions of non-coding RNAs have been discovered and their involvement in human disease, including cancer, became clear. In this review, we will present the evidence connecting modifications of different non-coding RNA subtypes and their role in cancer.

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

DSCAM-AS1 is a cancer-related long noncoding RNA with higher expression levels in Luminal A, B, and HER2-positive Breast Carcinoma (BC), where its expression is strongly dependent on Estrogen Receptor Alpha (ERα). DSCAM-AS1 expression is analyzed in 30 public datasets and, additionally, by qRT-PCR in tumors from 93 BC patients, to uncover correlations with clinical data. Moreover, the effect of DSCAM-AS1 knockdown on gene expression and alternative splicing is studied by RNA-Seq in MCF-7 cells. We confirm DSCAM-AS1 overexpression in high grade Luminal A, B, and HER2+ BCs and find a significant correlation with disease relapse. In total, 908 genes are regulated by DSCAM-AS1-silencing, primarily involved in the cell cycle and inflammatory response. Noteworthily, the analysis of alternative splicing and isoform regulation reveals 2085 splicing events regulated by DSCAM-AS1, enriched in alternative polyadenylation sites, 3'UTR (untranslated region) shortening and exon skipping events. Finally, the DSCAM-AS1-interacting splicing factor heterogeneous nuclear ribonucleoprotein L (hnRNPL) is predicted as the most enriched RBP for exon skipping and 3'UTR events. The relevance of DSCAM-AS1 overexpression in BC is confirmed by clinical data and further enhanced by its possible involvement in the regulation of RNA processing, which is emerging as one of the most important dysfunctions in cancer.

Docker4Circ: A Framework for the Reproducible Characterization of circRNAs from RNA-Seq Data.

Recent improvements in cost-effectiveness of high-throughput technologies has allowed RNA sequencing of total transcriptomes suitable for evaluating the expression and regulation of circRNAs, a relatively novel class of transcript isoforms with suggested roles in transcriptional and post-transcriptional gene expression regulation, as well as their possible use as biomarkers, due to their deregulation in various human diseases. A limited number of integrated workflows exists for prediction, characterization, and differential expression analysis of circRNAs, none of them complying with computational reproducibility requirements. We developed Docker4Circ for the complete analysis of circRNAs from RNA-Seq data. Docker4Circ runs a comprehensive analysis of circRNAs in human and model organisms, including: circRNAs prediction; classification and annotation using six public databases; back-splice sequence reconstruction; internal alternative splicing of circularizing exons; alignment-free circRNAs quantification from RNA-Seq reads; and differential expression analysis. Docker4Circ makes circRNAs analysis easier and more accessible thanks to: (i) its R interface; (ii) encapsulation of computational tasks into docker images; (iii) user-friendly Java GUI Interface availability; and (iv) no need of advanced bash scripting skills for correct use. Furthermore, Docker4Circ ensures a reproducible analysis since all its tasks are embedded into a docker image following the guidelines provided by Reproducible Bioinformatics Project.

The expression of LINE1-MET chimeric transcript identifies a subgroup of aggressive breast cancers.

Demethylation of the long interspersed nuclear element (LINE-1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1-MET transcript produced by the L1 placed in the second intron of MET. To define the role of L1-MET, we investigated the sequence and the transcription of L1-MET in vitro models and heterogeneous breast cancers, previously reported to show other L1-derived transcripts. L1-MET expressing cell lines were initially identified in silico and investigated for L1-MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1-MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1-MET transcript ending at MET exon 21, six novel L1-MET splice variants were identified. Normal breast tissues were negative for the L1-MET expression, whereas the triple-negative breast cancer (TNBC) and the high-grade carcinomas were enriched with the L1-MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1-MET expression was associated with its promoter hypomethylation (ρ = -0.8 and -0.9, respectively). No correlation was found between L1-MET and MET mRNA although L1-MET expressing tumors with higher L1-MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1-MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1-MET in the challenging diagnosis of early TNBCs.

Luminal breast cancer-specific circular RNAs uncovered by a novel tool for data analysis.

Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcript processing. We have exploited poly(A-) RNA-Seq data generated in our lab in MCF-7 breast cancer cells to define a compilation of exonic circRNAs more comprehensive than previously existing lists. Development of a novel computational tool, named CircHunter, allowed us to more accurately characterize circRNAs and to quantitatively evaluate their expression in publicly available RNA-Seq data from breast cancer cell lines and tumor tissues. We observed and confirmed, by ChIP analysis, that exons involved in circularization events display significantly higher levels of the histone post-transcriptional modification H3K36me3 than non-circularizing exons. This result has potential impact on circRNA biogenesis since H3K36me3 has been involved in alternative splicing mechanisms. By analyzing an Ago-HITS-CLIP dataset we also found that circularizing exons overlapped with an unexpectedly higher number of Ago binding sites than non-circularizing exons. Finally, we observed that a subset of MCF-7 circRNAs are specific to tumor versus normal tissue, while others can distinguish Luminal from other tumor subtypes, thus suggesting that circRNAs can be exploited as novel biomarkers and drug targets for breast cancer.

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

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.

Dissecting the genomic activity of a transcriptional regulator by the integrative analysis of omics data.

In the study of genomic regulation, strategies to integrate the data produced by Next Generation Sequencing (NGS)-based technologies in a meaningful ensemble are eagerly awaited and must continuously evolve. Here, we describe an integrative strategy for the analysis of data generated by chromatin immunoprecipitation followed by NGS which combines algorithms for data overlap, normalization and epigenetic state analysis. The performance of our strategy is illustrated by presenting the analysis of data relative to the transcriptional regulator Estrogen Receptor alpha (ERα) in MCF-7 breast cancer cells and of Glucocorticoid Receptor (GR) in A549 lung cancer cells. We went through the definition of reference cistromes for different experimental contexts, the integration of data relative to co-regulators and the overlay of chromatin states as defined by epigenetic marks in MCF-7 cells. With our strategy, we identified novel features of estrogen-independent ERα activity, including FoxM1 interaction, eRNAs transcription and a peculiar ontology of connected genes.

A Novel Functional Domain of Tab2 Involved in the Interaction with Estrogen Receptor Alpha in Breast Cancer Cells.

Tab2, originally described as a component of the inflammatory pathway, has been implicated in phenomena of gene de-repression in several contexts, due to its ability to interact with the NCoR corepressor. Tab2 interacts also with steroid receptors and dismisses NCoR from antagonist-bound Estrogen and Androgen Receptors on gene regulatory regions, thus modifying their transcriptional activity and leading to pharmacological resistance in breast and prostate cancer cells. We demonstrated previously that either Tab2 knock-down, or a peptide mimicking the Estrogen Receptor alpha domain interacting with Tab2, restore the antiproliferative response to Tamoxifen in Tamoxifen-resistant breast cancer cells. In this work, we map the domain of Tab2 responsible of Estrogen Receptor alpha interaction. First, using both co-immunoprecipitation and pull-down with recombinant proteins, we found that the central part of Tab2 is primarily responsible for this interaction, and that this region also interacts with Androgen Receptor. Then, we narrowed down the essential interaction region by means of competition assays using recombinant protein pull-down. The interaction motif was finally identified as a small region adjacent to, but not overlapping, the Tab2 MEKK1 phosphorylation sites. A synthetic peptide mimicking this motif efficiently displaced Tab2 from interacting with recombinant Estrogen Receptor alpha in vitro, prompting us to test its efficacy using derivatives of the MCF7 breast carcinoma cell lines that are spontaneously resistant to Tamoxifen. Indeed, we observed that this mimic peptide, made cell-permeable by addition of the TAT minimal carrier domain, reduced the growth of Tamoxifen-resistant MCF7 cells in the presence of Tamoxifen. These data indicate a novel functional domain of the Tab2 protein with potential application in drug design.

E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells.

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERß) that bind to specific regulatory genomic regions. We focused our attention on 17ß-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer.

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.