|Replacement Information||04-820 is a recommended replacement for 05-820|
Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R, B, Po||WB, IP||Rb||Culture Supernatant||Monoclonal Antibody|
|Presentation||Cultured supernantant with 0.05% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Stable for 1 year at -20°C from date of receipt.
For maximum recovery of product, centrifuge the vial prior to removing the cap.
|Material Size||100 µL|
|Reference overview||Application||Pub Med ID|
|Resistance to everolimus driven by epigenetic regulation of MYC in ER+ breast cancers. |
Bihani, T; Ezell, SA; Ladd, B; Grosskurth, SE; Mazzola, AM; Pietras, M; Reimer, C; Zinda, M; Fawell, S; D'Cruz, CM
Oncotarget 6 2407-20 2015
Acquired resistance to PI3K/mTOR/Akt pathway inhibitors is often associated with compensatory feedback loops involving the activation of oncogenes. Here, we have generated everolimus resistance in ER+ breast cancer cells and in long-term estrogen deprived (LTED) models that mimic progression on anti-estrogens. This allowed us to uncover MYC as a driver of mTOR inhibitor resistance. We demonstrate that both everolimus resistance and acute treatment of everolimus can lead to the upregulation of MYC mRNA, protein expression and, consequently, the enrichment of MYC signatures as revealed by RNA sequencing data. Depletion of MYC resulted in resensitization to everolimus, confirming its functional importance in this setting. Furthermore, ChIP assays demonstrate that MYC upregulation in the everolimus resistant lines is mediated by increased association of the BRD4 transcription factor with the MYC gene. Finally, JQ1, a BRD4 inhibitor combined with everolimus exhibited increased tumor growth inhibition in 3D Matrigel models and an in vivo xenograft model. These data suggest that MYC plays an important role in mediating resistance to everolimus in ER+ and ER+/LTED models. Furthermore, given the regulation ofMYCby BRD4 in this setting, these data have implications for increased therapeutic potential of combining epigenetic agents with mTOR inhibitors to effectively downregulate otherwise difficult to target transcription factors such as MYC.
|Unraveling the regulatory connections between two controllers of breast cancer cell fate. |
Lee, J; Tiwari, A; Shum, V; Mills, GB; Mancini, MA; Igoshin, OA; Balázsi, G
Nucleic acids research 42 6839-49 2014
Estrogen receptor alpha (ERα) expression is critical for breast cancer classification, high ERα expression being associated with better prognosis. ERα levels strongly correlate with that of GATA binding protein 3 (GATA3), a major regulator of ERα expression. However, the mechanistic details of ERα-GATA3 regulation remain incompletely understood. Here we combine mathematical modeling with perturbation experiments to unravel the nature of regulatory connections in the ERα-GATA3 network. Through cell population-average, single-cell and single-nucleus measurements, we show that the cross-regulation between ERα and GATA3 amounts to overall negative feedback. Further, mathematical modeling reveals that GATA3 positively regulates its own expression and that ERα autoregulation is most likely absent. Lastly, we show that the two cross-regulatory connections in the ERα-GATA3 negative feedback network decrease the noise in ERα or GATA3 expression. This may ensure robust cell fate maintenance in the face of intracellular and environmental fluctuations, contributing to tissue homeostasis in normal conditions, but also to the maintenance of pathogenic states during cancer progression.
|Fluorescence Activated Cell Sorting (FACS)||24792166|
|Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays. |
Stossi, F; Bolt, MJ; Ashcroft, FJ; Lamerdin, JE; Melnick, JS; Powell, RT; Dandekar, RD; Mancini, MG; Walker, CL; Westwick, JK; Mancini, MA
Chemistry & biology 21 743-53 2014
Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERβ loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERβ and act as ERβ antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.
|JMJD6 regulates ERα methylation on arginine. |
Poulard, C; Rambaud, J; Hussein, N; Corbo, L; Le Romancer, M
PloS one 9 e87982 2014
ERα functions are tightly controlled by numerous post-translational modifications including arginine methylation, which is required to mediate the extranuclear functions of the receptor. We report that upon oestrogenic stimulation, JMJD6, the only arginine demethylase described so far, interacts with and regulates methylated ERα (metERα) function. Moreover, by combining the silencing of JMJD6 with demethylation assays, we show that metERα is a new substrate for JMJD6. We propose that the demethylase activity of JMJD6 is a decisive regulator of the rapid physiological responses to oestrogen.
|Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses. |
Bolt, MJ; Stossi, F; Newberg, JY; Orjalo, A; Johansson, HE; Mancini, MA
Nucleic acids research 41 4036-48 2013
Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other's activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.
|FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection. |
Johansen, LM; Brannan, JM; Delos, SE; Shoemaker, CJ; Stossel, A; Lear, C; Hoffstrom, BG; Dewald, LE; Schornberg, KL; Scully, C; Lehár, J; Hensley, LE; White, JM; Olinger, GG
Science translational medicine 5 190ra79 2013
Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)- and ex-US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.
|Activation of rapid oestrogen signalling in aggressive human breast cancers. |
Poulard, C; Treilleux, I; Lavergne, E; Bouchekioua-Bouzaghou, K; Goddard-Léon, S; Chabaud, S; Trédan, O; Corbo, L; Le Romancer, M
EMBO molecular medicine 4 1200-13 2012
Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.
|High content imaging-based assay to classify estrogen receptor-α ligands based on defined mechanistic outcomes. |
Ashcroft, FJ; Newberg, JY; Jones, ED; Mikic, I; Mancini, MA
Gene 477 42-52 2011
Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.
|Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing. |
Billon-Galés, A; Krust, A; Fontaine, C; Abot, A; Flouriot, G; Toutain, C; Berges, H; Gadeau, AP; Lenfant, F; Gourdy, P; Chambon, P; Arnal, JF
Proceedings of the National Academy of Sciences of the United States of America 108 13311-6 2011
17β-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2(0)). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2(0) to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2(0) mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2(0) in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2(0) LDL-r(-/-) mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.
|ATBF1 inhibits estrogen receptor (ER) function by selectively competing with AIB1 for binding to the ER in ER-positive breast cancer cells. |
Dong, Xue-Yuan, et al.
J. Biol. Chem., 285: 32801-9 (2010) 2010
Loss of the q22 band of chromosome 16 is a frequent genetic event in breast cancer, and the candidate tumor suppressor gene, ATBF1, has been implicated in breast cancer by genomic deletion, transcriptional down-regulation, and association with better prognostic parameters. In addition, estrogen receptor (ER)-positive breast cancer expresses a higher level of ATBF1, suggesting a role of ATBF1 in ER-positive breast cancer. In this study, we examined whether and how ATBF1 affects the ER function in breast cancer cells. We found that ATBF1 inhibited ER-mediated gene transcription, cell growth, and proliferation in ER-positive breast cancer cells. In vitro and in vivo immunoprecipitation experiments revealed that ATBF1 interacted physically with the ER and that multiple domains in both ATBF1 and ER proteins mediated the interaction. Furthermore, we demonstrated that ATBF1 inhibited ER function by selectively competing with the steroid receptor coactivator AIB1 but not GRIP1 or SRC1 for binding to the ER. These findings not only support the concept that ATBF1 plays a tumor-suppressive role in breast cancer, they also provide a mechanism for how ATBF1 functions as a tumor suppressor in breast cancer.