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.

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

Estrogen receptors α (ER-α) and ß (ER-ß) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-ß 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-ß 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-ß interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-ß 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.

Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells.

BACKGROUND: BPA (bisphenol A or 2,2-bis(4-hydroxy-phenol)propane) is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound. METHODS: Cell cycle, apoptosis and differentiation analyses; western blots. RESULTS: BPA is able to induce cell cycle arrest and apoptosis in three different acute myeloid leukemias. Although some granulocytic differentiation concomitantly occurred in NB4 cells upon BPA treatment, the major action was the induction of apoptosis. BPA mediated apoptosis was caspase dependent and occurred by activation of extrinsic and intrinsic cell death pathways modulating both FAS and TRAIL and by inducing BAD phosphorylation in NB4 cells. Finally, also non genomic actions such as the early decrease of both ERK and AKT phosphorylation were induced by BPA thus indicating that a complex intersection of regulations occur for the apoptotic action of BPA. CONCLUSION: BPA is able to induce apoptosis in leukemia cells via caspase activation and involvement of both intrinsic and extrinsic pathways of apoptosis.

Quantitative expression profiling of highly degraded RNA from formalin-fixed, paraffin-embedded breast tumor biopsies by oligonucleotide microarrays.

Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, particularly formalin-fixed, paraffin-embedded (FFPE) archived tissues, is limited by the poor quality of the RNA recovered. This represents a serious drawback, as FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer (MT) biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17beta-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.

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 17ß-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.

Comparative analysis of nuclear estrogen receptor alpha and beta interactomes in breast cancer cells.

Estrogen Receptor alpha and beta (ER-α and -ß) are members of the nuclear receptor family of transcriptional regulators with distinct roles in mediating estrogen dependent breast cancer cell growth and differentiation. Following activation by the hormone, these proteins undergo conformation changes and accumulate in the nucleus, where they bind to chromatin at regulatory sites as homo- and/or heterodimers and assemble in large multiprotein complexes. Although the two ERs share a conserved structure, they exert specific and distinct functional roles in normal and transformed mammary epithelial cells and other cell types. To investigate the molecular bases of such differences, we performed a comparative computational analysis of the nuclear interactomes of the two ER subtypes, exploiting two datasets of receptor interacting proteins identified in breast cancer cell nuclei by Tandem Affinity Purification for their ability to associate in vivo with ligand-activated ER-α and/or ER-ß. These datasets comprise 498 proteins, of which only 70 are common to both ERs, suggesting that differences in the nature of the two ER interactomes are likely to sustain the distinct roles of the two receptor subtypes. Functional characterization of the two interactomes and their topological analysis, considering node degree and closeness of the networks, confirmed this possibility. Indeed, clustering and network dissection highlighted the presence of distinct and ER subtype-specific subnetworks endowed with defined functions. Altogether, these data provide new insights on the protein-protein interaction networks controlled by ER-α and -ß that mediate their ability to transduce estrogen signaling in breast cancer cells.