RESUMEN
Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process modulated by coactivator and corepressor proteins. Breast Cancer Amplified Sequence 2 (BCAS2), is a poorly studied ERα coactivator. In this work, we characterize some of the mechanisms through which this protein increases ERα activity and how this promotes carcinogenic processes in breast cancer cells. Using protein-protein interaction and luciferase assays we show that BCAS2 interacts with ERα both in vitro and in vivo and upregulates transcriptional activation of ERα directly through its N-terminal region (AF-1) and indirectly through its C-terminal (AF-2) region, acting in concert with AF-2 interacting coactivators. Elevated expression of BCAS2 positively affects proliferation, clonogenicity and migration of breast cancer cells and directly activates ERα regulated genes which have been shown to play a role in tumor growth and progression. Finally, we used signal transduction pathway inhibitors to elucidate how BCAS2 is regulated in these cells and observed that BCAS2 is preferentially regulated by the PI3K/AKT signaling pathway. BCAS2 is an AF-1 coactivator of ERα whose overexpression promotes carcinogenic processes, suggesting an important role in the development of estrogen-receptor positive breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Receptor alfa de Estrógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/química , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/química , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
More than 70% of all breast cancer cases are estrogen receptor alpha-positive (ERα). ERα is a member of the nuclear receptor family, and its activity is implicated in the gene transcription linked to the proliferation of breast cancer cells, as well as in extranuclear signaling pathways related to the development of resistance to endocrine therapy. Protein-protein interactions and posttranslational modifications of ERα underlie critical mechanisms that modulate its activity. In this review, the relationship between ERα and ubiquitin protein (Ub), was investigated in the context of breast cancer cells. Interestingly, Ub can bind covalently or non-covalently to ERα resulting in either a proteolytic or non-proteolytic fate for this receptor. Thereby, Ub-dependent molecular pathways that modulate ERα signaling may play a central role in breast cancer progression, and consequently, present critical targets for treatment of this disease.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Ubiquitina/metabolismo , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Unión Proteica , Proteolisis , Transducción de Señal , UbiquitinaciónRESUMEN
Estrogen receptor alpha (ERα) has an established role in breast cancer biology. Transcriptional activation by ERα is a multistep process influenced by coactivator and corepressor proteins. This work shows that Pin2 interacting protein 1 (PINX1) interacts with the N-terminal domain of ERα and functions as a corepressor of ERα. Furthermore, it represses both AF-1 and AF-2 transcriptional activities. Chromatin immunoprecipitation assays verified that the interaction between ERα and PINX1 occurs on E2 regulated promoters and enhanced expression of PINX1 deregulates the expression of a number of genes that have a role in cell growth and proliferation in breast cancer. PINX1 overexpression decreases estrogen mediated proliferation of breast cancer cell lines, while its depletion shows the opposite effect. Taken together, these data show a novel molecular mechanism for PINX1 as an attenuator of estrogen receptor activity in breast cancer cell lines, furthering its role as a tumor suppressor gene in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Células COS , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Chlorocebus aethiops , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Células MCF-7 , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genéticaRESUMEN
Salpichrolides are natural plant steroids that contain an unusual six-membered aromatic ring D. We recently reported that some of these compounds, and certain analogs with a simplified side chain, exhibited antagonist effects toward the human estrogen receptor (ER), a nuclear receptor whose endogenous ligand has an aromatic A ring (estradiol). Drugs acting through the inhibition or modulation of ERs are frequently used as a hormonal therapy for ER(+) breast cancer. Previous results suggested that the aromatic D ring was a key structural motif for the observed activity; thus, this modified steroid nucleus may provide a new scaffold for the design of novel antiestrogens. Using molecular dynamics (MD) simulation we have modeled the binding mode of the natural salpichrolide A and a synthetic analog with an aromatic D ring within the ERα. These results taken together with the calculated energetic contributions associated to the different ligand-binding modes are consistent with a preferred inverted orientation of the steroids in the ligand-binding pocket with the aromatic ring D occupying a position similar to that observed for the A ring of estradiol. Major changes in both dynamical behavior and global positioning of H11 caused by the loss of the ligand-His524 interaction might explain, at least in part, the molecular basis of the antagonism exhibited by these compounds. Using steered MD we also found a putative unbinding pathway for the steroidal ligands through a cavity formed by residues in H3, H7, and H11, which requires only minor changes in the overall receptor conformation.
Asunto(s)
Ergosterol/análogos & derivados , Estradiol/química , Moduladores de los Receptores de Estrógeno/química , Receptor alfa de Estrógeno/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Secuencias de Aminoácidos , Sitios de Unión , Ergosterol/síntesis química , Ergosterol/química , Moduladores de los Receptores de Estrógeno/síntesis química , Receptor alfa de Estrógeno/química , Humanos , Ligandos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica , Interfaz Usuario-ComputadorRESUMEN
A learning module for molecular level analysis of protein structure and ligand/drug interaction through the visualization of X-ray diffraction is presented. Using DeepView as molecular model visualization software, students learn about the general concepts of protein structure. This Biochemistry classroom exercise is designed to be carried out by following the detailed instructions that make software handling straightforward. Students learn about protein structure and gain insight into the molecular level of the interaction of two active compounds with their receptor. © 2013 by The International Union of Biochemistry and Molecular Biology 41(2):118-124, 2013.
Asunto(s)
Bioquímica/educación , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Programas Informáticos , Estradiol/química , Estradiol/metabolismo , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Clorhidrato de Raloxifeno/química , Clorhidrato de Raloxifeno/metabolismo , EstudiantesRESUMEN
Four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis was applied on a series of 54 2-arylbenzothiophene derivatives, synthesized by Grese and coworkers, based on raloxifene (an estrogen receptor-alpha antagonist), and evaluated as ERa ligands and as inhibitors of estrogen-stimulated proliferation of MCF-7 breast cancer cells. The conformations of each analogue, sampled from a molecular dynamics simulation, were placed in a grid cell lattice according to three trial alignments, considering two grid cell sizes (1.0 and 2.0 Å). The QSAR equations, generated by a combined scheme of genetic algorithms (GA) and partial least squares (PLS) regression, were evaluated by "leave-one-out" cross-validation, using a training set of 41 compounds. External validation was performed using a test set of 13 compounds. The obtained 4D-QSAR models are in agreement with the proposed mechanism of action for raloxifene. This study allowed a quantitative prediction of compounds' potency and supported the design of new raloxifene analogs.
Asunto(s)
Relación Estructura-Actividad Cuantitativa , Clorhidrato de Raloxifeno/análogos & derivados , Moduladores Selectivos de los Receptores de Estrógeno/química , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/química , Concentración 50 Inhibidora , Conformación Molecular , Simulación de Dinámica Molecular , Unión Proteica , Clorhidrato de Raloxifeno/química , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacologíaRESUMEN
Compounds with estrogenic effects that also inhibit platelet aggregation might be useful in reducing thrombotic events associated with estrogenic therapy. In this study, two aminoestrogens, Buame [N-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-butylamine] and Diebud [N,N'-bis-(3-hydroxy-1,3,5(10)-estratrien-17ß-yl)-1,4-butanediamine], were synthesized and characterized using common analytical methods and spectrophotometric analyses. The location and orientation of these molecules on the estrogenic receptor α (ERα) were also evaluated. Platelet inhibitory effects were elucidated ADP-induced platelet aggregation and ADP- and collagen-induced ATP release. Molecular docking demonstrated that Buame can reach and bind to the ERα in the ligand binding domain (LBD) similar to 17ß-estradiol (co-crystallized ligand). On the other hand, Diebud binds only to the surface of ERα due to its high molecular volume compared to 17ß-estradiol and Buame.
Asunto(s)
Congéneres del Estradiol/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Adulto , Sitios de Unión , Colágeno/farmacología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Congéneres del Estradiol/química , Congéneres del Estradiol/metabolismo , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Humanos , Liposomas/química , Masculino , Persona de Mediana Edad , Modelos Moleculares , Estructura Molecular , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Adulto JovenRESUMEN
The effects of estradiol on the Harderian gland (HG) are believed to be partially regulated by the transcriptional regulation of the estrogen-related genes via estrogen receptor (ER). In reptiles, however, it has not been well established whether the HG contains or expresses steroid nuclear receptors. As a first step toward investigating the molecular mechanisms of estrogen signalling in the HG, we isolated the cDNA for ERalpha in the sea turtle Lepidochelys olivacea. ERalpha was cloned using RT-PCR coupled with 5' and 3' RACE procedures. The cDNA contains a complete open reading frame encoding 588 amino acid residues. Comparative analysis of this amino acid sequence showed moderate to strong conservation of the ERalpha (Esr1) gene within divergent vertebrate groups. In transfection studies, the cloned ER displayed high affinity K(d)=0.25nM and high specificity for 17beta-estradiol. Binding assays using sucrose density gradients demonstrated a specific 7-7.5 S binding component in the HG cytosolic fractions. RT-qPCR analysis showed significant ERalpha mRNA expression in the liver, HG, lung and brain. Altogether, these results provide evidence for the expression of intracellular ERs in the HG of the sea turtle and suggest that ERalpha may be an important modulator of the estrogen-mediated response in the HG of reptiles.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Proteínas de Reptiles/metabolismo , Tortugas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/genética , Humanos , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas de Reptiles/química , Proteínas de Reptiles/genética , Alineación de Secuencia , Análisis de Secuencia , Transfección , Tortugas/genéticaRESUMEN
We have studied the modulation and differential sensitivity of the estrogen receptor (ER) in breast cancer in the presence of protecting or modifying agents of disulfide bonds and sulfhydryl groups present in the ER steroid-binding domain. Protecting agents such as mercaptoethanol and dithiotreitol increased the [(3)H]estradiol binding to ER by 25% and 50%, respectively. Modifying agents such as p-chloromercuribenzensulfonate decreased the [(3)H]estradiol binding by 70% and this was nearly completely abolished by N-ethylmaleimide (94%). These data indicate that disulfide bonds and sulfhydryl groups in the steroid-binding domain are intimately involved in the maintenance of a receptor structure necessary for estradiol binding.
Asunto(s)
Neoplasias de la Mama/química , Disulfuros/análisis , Receptor alfa de Estrógeno/química , Compuestos de Sulfhidrilo/análisis , Neoplasias de la Mama/fisiopatología , Estradiol/metabolismo , Femenino , Humanos , Relación Estructura-ActividadRESUMEN
As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.
Asunto(s)
Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/ultraestructura , Modificación Traduccional de las Proteínas , Sitios de Unión , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Isoformas de Proteínas/químicaRESUMEN
BACKGROUND: Estrogens exert profound effects on target tissues. These effects are mediated by two estrogen receptors (ER(alpha) and ER(beta)) that bind to specific DNA sequences in estrogen-dependent genes. Other molecules such as growth factors, transcription factors and some oncoproteins might interact with the estrogen receptors and thus regulate the transcription of these genes. Currently there is no adequate cellular model to study these interactions. METHODS: We transfected the human wild-type ER(alpha) to an ER-negative rat epithelial endometrial cell line (Rentr01) using a tetracycline-regulated gene expression system. The exogenous receptor was correctly translated, had an appropriate hormone-binding affinity, and bound well to estrogen response elements containing DNA. RESULTS: We obtained a new stable cell line that is ER(beta) negative but ER(alpha) positive (R1-49E1). The expression of receptor alpha can be regulated in a dose-response manner by addition of tetracycline in the culture medium. Estradiol treatment of ER(alpha)-containing cells apparently diminished cellular proliferation, and the exogenous receptor can induce the transcription of the endogenous progesterone receptor isoform B (PgR-B) gene. CONCLUSIONS: This epithelial cellular model may be useful to study the interaction between estrogens and other cell signaling pathways in epithelial endometrial cell physiology.
Asunto(s)
Línea Celular , Endometrio/citología , Receptor alfa de Estrógeno/biosíntesis , Tetraciclina/farmacología , Animales , Diferenciación Celular , Proliferación Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , Medios de Cultivo/farmacología , ADN/química , Relación Dosis-Respuesta a Droga , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Células 3T3 NIH , Unión Proteica , Ratas , Receptores de Progesterona/metabolismo , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Fibroadenomas are the most common benign lump in females. The study of gene alterations and/or deregulation in reproductive years may help explain hormonal physiological processes involved in nodule development and evolution. The objective was to compare ER-alpha, c-myc, and bcl-2 gene expression in breast fibroadenomas and in normal tissue and evaluate menstrual cycle, parity, and oral contraceptive influences. Fifty-seven premenopausal women (14-49 years) undergoing surgical removal of fibroadenomas were selected. Samples from fibroadenomas and circumjacent normal tissue were obtained for RT-PCR paired analysis. Patients were divided in groups according to menstrual cycle, use of contraceptives and parity. Tissue from 32 patients was adequate for RT-PCR. Paired analysis showed higher expression of ER-alpha (P=0.012) and bcl-2 (P=0.001) in fibroadenomas than in normal breast, while c-myc presented a similar expression (P=0.655). ER-alpha was higher in fibroadenomas of patients in follicular phase versus contraceptive users and normal tissue (P=0.003); bcl-2 was higher in fibroadenomas of patients in luteal phase than in the normal samples from all groups (P=0.007). c-myc did not differ according to menstrual cycle, but was higher in fibroadenomas>3 cm versus<3 cm (P=0.015) and in nulliparous women (P=0.04). A positive correlation between c-myc levels and fibroadenoma diameter was demonstrated (r=0.536; P=0.007). Nulliparous mean nodule diameter was superior than parous women (P=0.008). In conclusion, the expression of ER-alpha, bcl-2 and c-myc depends on hormonal and reproductive factors, with a possible contribution to lump formation and evolution.