Luminal-like breast tumor cells express estrogen receptor ␣ (ER␣),

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.

Estrogen receptor ␣ (ER␣) 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, ER␣ 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 hormoneresponsive genes. Identification of the molecular partners of ER␣ 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 ER␣ interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ER␣ fused to a tandem affinity purification tag were generated and used to purify native nuclear ERcontaining 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 liganddependent multiprotein complex comprising ␤-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 ER␣ 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 ER␣ actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis. Molecular & Cellular Proteomics 9:1352-1367, 2010.Estrogens are potent tumor promoters for the mammary gland due to their growth-promoting actions in mammary epithelial cells (1). The mechanisms underlying stimulation of breast cell proliferation and control of the cell state by estrogens are still poorly defined despite the evident causal relationships between these hormonal actions and mammary gland carcinogenesis and cancer progression. Estrogen-responsive cells are endowed with specific estrogen receptors, ER␣ 1 and ER␤, members of the steroid/nuclear receptor superfamily of transcription factors that directly modulate the gene transcription rate (2). In addition, estrogens can trigger rapid and transient cellular responses through a mechanism(s) independent from this "genomic" pathway of steroid receptor action (3, 4). Such "extragenomic" effects include cell typespecific, rapid, and transient responses of signal transduction pathways; induction of intracellular calcium mobilization; and From the Departmen...