Francesca Cirillo

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.

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.

Associated vascular lesions in patients undergoing coronary artery bypass grafting

Atherosclerotic involvement of extracoronary arteries in patients undergoing myocardial revascularization can cause severe postoperative complications and increase postoperative mortality. Between January and November 1998, routine preoperative echo-Doppler study of carotid vessels, abdominal aorta and iliac-femoral arteries was performed in all patients undergoing coronary artery bypass grafting (CABG) at our institution, in order to assess the prevalence and the degree of associated vascular lesions. Correlations between echo-Doppler findings, angiographic patterns of coronary lesions and atherosclerotic risk factors were analyzed in all cases. Among 302 patients undergoing CABG, 186 (61.6%) had carotid disease, with a haemodynamically significant stenosis (>70%) of internal carotid in 31 (10.2%).Twenty-three patients had asymptomatic severe carotid disease. A significant correlation between severity of coronary disease and prevalence of severe carotid disease was found (p = 0.02). An abdominal aortic dilatation (diameter > 25 mm) was found in 20 cases (6.6%), with a diameter >35 mm in 7 patients (2.3%), 6 with triple-vessel coronary disease, and 1 with double-vessel disease. Atherosclerotic lesions of iliac-femoro-popliteal axis were found in 165 (54.6%) patients, with a strong correlation to the severity of coronary disease (p = 0.02); lesions were haemodynamically significant (> 70%) in 48 (15.8%) cases. Symptoms of carotid and peripheral vascular disease are no reliable predictors of perioperative risk in patients undergoing CABG. Non-invasive complete arterial investigation should be routinely performed in these patients, in order to plan the most suitable operative approach and to prevent perioperative vascular complications.

New Insights on Estrogen Receptor Actions in Hormone-Responsive Breast Cancer Cells by Interaction Proteomics

Estrogens are tumor promoters for the mammary gland, due to their ability to control multiple functions of target cells and to stimulate their proliferation. The mechanisms that underlie control of cell proliferation by estrogens are still not fully defined, despite the important causal relationships between this hormonal action, mammary gland carcinogenesis, and breast cancer (BC) progression. Estrogens exert their actions in target tissues via two intracellular receptors, ERalpha (ERα) and ERbeta (ERβ), that show specific, and often antagonist, roles and can be found co-expressed in BC where, however, ERα appears to prevail in mediating estrogen actions. ERs are ligand-dependent transcription factors of the nuclear receptor superfamily of intracellular regulators, and their activity is tightly controlled by hormonal and non-hormonal ligands. This notion led to the design of synthetic ER antagonist ligands, including steroidal and nonsteroidal antiestrogens, that are effective to inhibit BC cell proliferation and, for this reason, widely used for treatment of hormone-responsive tumors. These drugs, however, exhibit side effects that limit their efficacy and use. Studies based on application of genomics and proteomics are revealing new insights on estrogen signaling in BC cells, with the discovery of novel ER partner proteins promising as potential novel drug targets. We review here the new insights on ER signaling derived by systematic application of interaction proteomics to map and characterize the intracellular network of proteins binding to agonist- and/or antagonist-activated ERα in a hormone-responsive human BC cell model.