Giovanni Nassa

Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

BackgroundEstrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology.ResultsExpression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions.ConclusionsResults indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERβ, transcription factors that display functional antagonism with each other, with ERβ acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERβ, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERβ might influence BC cell behavior via miRNAs, we compared miRNome expression in ERβ+ vs ERβ− hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERβ in BC cells, clearly distinguishes ERβ+, node-negative, from ERβ−, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERβ+ in BC cell nuclei. In particular, ERβ downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERβ in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.

Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei

Estrogen receptor α (ERα) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERα assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERα and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERα interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERα fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising β-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERα and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERα actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.

Effects of Oestrogen on MicroRNA Expression in Hormone-Responsive Breast Cancer Cells

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.

iMir: An integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq

BackgroundQualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once.ResultsWe designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of differentially expressed miRNAs. In addition, iMir allowed also the identification of ~70 piRNAs (piwi-interacting RNAs), some of which differentially expressed in proliferating vs growth arrested cells.ConclusionThe integrated data analysis pipeline described here is based on a reliable, flexible and fully automated workflow, useful to rapidly and efficiently analyze high-throughput smallRNA-Seq data, such as those produced by the most recent high-performance next generation sequencers. iMir is available at http://www.labmedmolge.unisa.it/inglese/research/imir.

A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei.

Estrogen receptors α (ER‐α) and β (ER‐β) play distinct biological roles in onset and progression of hormone‐responsive breast cancer, with ER‐β exerting a modulatory activity on ER‐α‐mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER‐β fused to a tandem affinity purification‐tag in estrogen‐responsive MCF‐7 cells and applied tandem affinity purification and nanoLC‐MS/MS to identify the ER‐β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co‐purify with ER‐β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.

Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS

Estrogen receptor α (ER‐α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand‐activated ER‐α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand‐activated ER‐α in MCF‐7 cell nuclei. This led to the identification of 264 ER‐associated proteins, whose functions highlight the hinge role of ER‐α in the coordination of multiple hormone‐regulated nuclear processes in BC cells.

Molecular mechanisms of selective estrogen receptor modulator activity in human breast cancer cells: identification of novel nuclear cofactors of antiestrogen-ERα complexes by interaction proteomics.

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.

Timed regulation of P‐element‐induced wimpy testis–interacting RNA expression during rat liver regeneration

Small noncoding RNAs comprise a growing family of molecules that regulate key cellular processes, including messenger RNA (mRNA) degradation, translational repression, and transcriptional gene silencing. P‐element‐induced wimpy testis (PIWI)‐interacting RNAs (piRNAs) represent a class of small RNAs initially identified in the germline of a variety of species, where they contribute to maintenance of genome stability, and recently found expressed also in stem and somatic cells, where their role and responsiveness to physiopathological signals remain elusive. Here, we investigated piRNA expression in rat liver and its response to the stimuli exerted by regenerative proliferation of this organ. Quantitative polymerase chain reaction analysis identify in the liver the RNAs encoding PIWIL2/HILI, PIWIL4/HIWI2, and other components of the piRNA biogenesis pathways, suggesting that this is indeed functional. RNA sequencing before, during, and after the wave of cell proliferation that follows partial hepatectomy (PH) identified ∼1,400 mammalian germline piRNAs expressed in rat liver, including 72 showing timed changes in expression 24‐48 hours post‐PH, a timing that corresponds to cell transition through the S phase, returning to basal levels by 168 hours, when organ regeneration is completed and hepatocytes reach quiescence. Conclusion: The piRNA pathway is active in somatic cells of the liver and is subject to regulation during the pathophysiological process of organ regeneration, when these molecules are available to exert their regulatory functions on the cell genome and transcriptome, as demonstrated by the identification of several liver mRNAs representing candidate targets of these regulatory RNAs. (Hepatology 2014;60:798–806)

Post-transcriptional Regulation of Human Breast Cancer Cell Proteome by Unliganded Estrogen Receptor β via microRNAs

Estrogen receptor β (ERβ) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. In contrast to ERα, its closest homolog, ERβ shows significant estrogen-independent activities, including the ability to inhibit cell cycle progression and regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERβ in BC MCF-7 and ZR-75.1 cells by means of microRNA (miRNA) sequencing, we identified 30 miRNAs differentially expressed in ERβ+ versus ERβ− cells in the absence of ligand, including up-regulated oncosuppressor miRs such miR-30a. In addition, a significant fraction of >1,600 unique proteins identified in MCF-7 cells by iTRAQ quantitative proteomics were either increased or decreased by ERβ, revealing regulation of multiple cell pathways by ligand-free receptors. Transcriptome analysis showed that for a large number of proteins regulated by ERβ, the corresponding mRNAs are unaffected, including a large number of putative targets of ERβ-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERβ. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERβ in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration was significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERβ on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation might represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.