Ernesto Nola

Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol‐dependent growth, estradiol treatment of human mammary cancer MCF‐7 cells triggers rapid and transient activation of the mitogen‐activated (MAP) kinases, erk‐1 and erk‐2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras‐GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP‐kinase pathway in MCF‐7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild‐type 67 kDa estradiol receptor cDNA that Cos cells become estradiol‐responsive in terms of erk‐2 activity. This finding, together with the inhibition by the pure anti‐estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF‐7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c‐src show that the estradiol receptor activates c‐src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c‐src is an initial and integral part of the signaling events mediated by the estradiol receptor.

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 17β-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.

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.

Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine‐phosphorylated hormone binding form and in non‐phosphorylated, non‐hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non‐hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase‐inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P‐phosphorylated proteins with anti‐receptor antibody followed either by sucrose gradient centrifugation or SDS‐PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti‐oestrogen tamoxifen. Oestradiol‐receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new.

Serum anti-p53 antibodies in lung cancer: comparison with established tumor markers

As reported earlier, p53 antibodies are detected in the sera of patients with different types of cancer, including lung cancer. In contrast, in the serum of healthy subjects the presence of anti-p53 antibodies is extremely rare. We collected the venous blood samples of 109 patients affected with lung cancer (LC): 57 patients (46 M, 11 F) with non-small-cell carcinoma (NSCLC), 52 others (40 M, 12 F) with small-cell carcinoma (SCLC). Serum p53 antibodies were assayed using ELISA method and all positive sera were confirmed by Western-blot method. In addition, using IRMA methods we assayed serum CEA, TPA, CYFRA21-1 and NSE. Serum p53Ab are detectable (p53Ab-positive) in 35/109 (32.1%) patients with lung cancer. About 17/57 (29.8%) patients affected with NSCLC and 18/52 (34.6%) with SCLC were p53Ab-positive. CEA, TPA, CYFRA21-1 and NSE sensitivity in LC patients (NSCLC+SCLC) is 50.5%, 58.7%, 42.2%, 35.8%, respectively. The lower sensitivity (32.1%) of serum p53Ab is connected with the higher specificity and diagnostic accuracy (100% and 69%, respectively). Out of 35 patients p53Ab-positive, five (14.3%) exhibit only serum p53Ab, while serum values of the established tumor markers were lower than cut-off. Serum p53Ab assessment is a simple and a low-cost assay with a good specificity and diagnostic accuracy that in LC patients can be used at least in association with established tumor markers.

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.

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.

In vitro phosphorylation and hormone binding activation of the synthetic wild type human estradiol receptor

A tyrosine kinase purified from calf uterus activates the hormone binding of endogenous estradiol receptor (ER) predephosphorylated and preinactivated by a nuclear phosphotyrosine phosphatase. The kinase also activates and phosphorylates the human estradiol receptor HEO synthesized in vitro, which differs from the wild type receptor HEGO because a glycine is replaced by a valine at position 400. Moreover, the kinase activates and phosphorylates a deletion mutant of HEO which consists almost exclusively of the hormone binding domain. Using HEGO and HEO in parallel and measuring both binding activation and phosphorylation of ER we now observe that the wild type receptor is a good kinase substrate, slightly better than HEO. Furthermore, HEGO like the calf uterus receptor in the presence of estradiol, stimulates the kinase. From present findings it appears that ER and uterus tyrosine kinase are functionally associated and that this association is abolished by glycine to valine substitution at position 400 of ER.

Estrogen receptor of calf uterus: An easy and fast purification procedure

Abstract We describe our method of purification of the “native” form of estradiol receptor of calf uterus. The high speed supernatant, after addition of 5mM MgCl 2 . is incubated batchwise with agarose to which heparin has been covalently bound. Elution of the estradiol binding activity is obtained by heparin. The volume of the eluate is reduced to one-tenth the volume of the original cytosol but presence of heparin avoids the aggregation of the receptor. Immobilization of receptor on 17β-estradiol-17-hemisuccinylhexane-agarosc (a very simple derivative to prepare) is best carried out on column. The washing of the specific adsorbent is very critical and must be extensive. Elution is performed with the chaotropic salt, NaSCN, in presence of low concentration of estradiol. A third step is finally required to eliminate some residual contaminating proteins. Sephadex G-200 chromatography in low salt buffer gives good results. “Native” receptor maintains, even after complete purification, the tendency to aggregate in very large forms which do not penetrate the polyacrylamide gel in the absence of sodium dodecyl sulphate, and are eluted in the void volume of Sephadex G-200 columns and very often sediment at the bottom of sucrose gradients. Presence of cations, in our case MgCl 2 , during the purification procedure decreases the aggregation phenomenon.