Abstract C56: Characterization of PinX1 as a coregulator of steroid hormone receptors
0
Citation
0
Reference
10
Related Paper
Abstract:
Abstract Introduction: Deregulated expression of transcriptional coactivators and corepressors has been implicated in tamoxifen (TAM) resistance, especially in ER+ breast cancer patients. The AF-1 transcriptional activation domain in the N-terminus of ER has been implicated in the tissue specific agonistic effects of TAM. Thus, using the yeast two-hybrid system with the N-terminal region of ERα, we located the PINX1 gene which encodes for a telomerase inhibitor. Objective: In this work, we characterized PinX1 as a coregulator of ERα. Methods and Results: Using GST pull-down assays, we confirmed the direct interaction between PinX1 and the N-terminus of ERα, showing no interaction with the C-terminus either in the presence or absence of estradiol (E2). Transcriptional activation assays using luciferase reporter genes show that PinX1 is capable of increasing ERα transcriptional activity in breast cancer cell lines, at low concentrations, but increasing PinX1 exhibits a negative effect on the transcriptional activity of ERα in these cell lines. However, Pinx1 repressed AF-1 transcriptional activity, as well as AF-2 activity in the presence of E2. ChIP assays verified that the interaction between ERα and Pinx1 occurs on E2 regulated promoters after 2h of incubation with E2. Furthermore, PinX1 inhibits E2 induced proliferation of MCF7 cells, while inhibition of PinX1 by siRNA increases E2 induced proliferation. These results suggest a role for PinX1 as a correpresor of ERα. PinX1 also interacts with the androgen and progesterone receptors. However, we demonstrate that PinX1 is a coactivator of these receptors by luciferase assays, as well as increasing DHT induced proliferation of prostate cancer cell lines. Enhanced expression of PinX1 also deregulates the expression of a number of genes that have a role in cell growth and proliferation in breast cancer cell lines. Conclusions: Our data support a dual role for PinX1 as a correpresor of ERα and a coactivator of AR and PR. Additionally, it may be involved in functions regulating cell growth and proliferation in both breast and prostate cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C56.Keywords:
Hormone response element
Hormone response element
Estrogen receptor beta
Cooperative binding
Estrogen receptor alpha
Antiestrogen
Cooperativity
Cite
Citations (31)
Cite
Citations (0)
Differential Response of Estrogen Receptor Subtypes to 1,3-Diarylindene and 2,3-Diarylindene Ligands
Estrogen receptors (ERs) control transcription of genes important for normal human development and reproduction. The signaling networks are complex, and there is a need for a molecular level understanding of the roles of receptor subtypes ERα and ERβ in normal physiology and as therapeutic targets. We synthesized two series of ER ligands, based on a common indene scaffold, in an attempt to develop compounds that can selectively modulate ER-mediated transcription. The 3-ethyl-1,2-diarylindenes, utilizing an amide linker for the 1-aryl extension, bind weakly to the ERs. The 2,3-diarylindenes bind with high affinity to the ER subtypes and demonstrate a range of different biological activities, both in transcriptional reporter gene assays and inhibition of estradiol-stimulated proliferation of MCF-7 cells. Several ligands differentiate between ERα and ERβ subtypes at an estrogen response element (ERE), displaying various levels of partial to full agonist activity at ERα, while antagonizing estradiol action at ERβ.
Hormone response element
Estrogen receptor beta
Transcription
Estrogen receptor alpha
Cite
Citations (114)
The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.
Hormone response element
Estrogen receptor beta
Estrogen receptor alpha
Estrogen-related receptor gamma
Estrogen-related receptor alpha
Cite
Citations (610)
Human estrogen receptor-alpha (hERalpha) or -beta (hERbeta) transfected into Hep G2 or COS1 cells each responded to estrogen to increase transcription from an estrogen-responsive element (ERE)-driven reporter vector with similar fold induction through a classical mechanism involving direct receptor binding to DNA. ER antagonists inhibited this estrogen induction through both hERalpha and hERbeta, although raloxifene was more potent through ERalpha than ERbeta, and tamoxifen was more potent via ERbeta than ERalpha. We have shown previously that estrogen stimulated the human retinoic acid receptor-alpha-1 (hRARalpha-1) promoter through nonclassical EREs by a mechanism that was ERalpha dependent, but that did not involve direct receptor binding to DNA. We show here that in contrast to hERalpha, hERbeta did not induce reporter activity driven by the hRARalpha-1 promoter in the presence of estrogen. While hERbeta did not confer estrogen responsiveness on this promoter, it did elicit transcriptional activation in the presence of 4-hydroxytamoxifen (4-OH-Tam). Additionally, this 4-OH-Tam agonist activity via ERbeta was completely blocked by estrogen. Like ERalpha, transcriptional activation of this promoter by ERbeta was not mediated by direct receptor binding to DNA. While hERalpha was shown to act through two estrogen-responsive sequences within the promoter, hERbeta acted only at the 3'-region, through two Sp1 sites, in response to 4-OH-Tam. Other ER antagonists including raloxifene, ICI-164,384 and ICI-182,780 also acted as agonists through ERbeta via the hRARalpha-1 promoter. Through the use of mutant and chimeric receptors, it was shown that the 4-OH-Tam activity via ERbeta from the hRARalpha-1 promoter in Hep G2 cells required the amino-terminal region of ERbeta, a region that was not necessary for estrogen-induced ERbeta activity from an ERE in Hep G2 cells. Additionally, the progesterone receptor (PR) antagonist RU486 acted as a weak (IC50 >1 microM) antagonist via hERalpha and as a fairly potent (IC50 approximately 200 nM) antagonist via hERbeta from an ERE-driven reporter in cells that do not express PR. Although RU486 bound only weakly to ERalpha or ERbeta in vitro, it did bind to ERbeta in whole-cell binding assays, and therefore, it is likely metabolized to an ERbeta-interacting compound in the cell. Interestingly, RU486 acted as an agonist through ERbeta to stimulate the hRARalpha-1 promoter in Hep G2 cells. These findings may have ramifications in breast cancer treatment regimens utilizing tamoxifen or other ER antagonists and may explain some of the known estrogenic or antiestrogenic biological actions of RU486.
Estrogen receptor beta
Hormone response element
Estrogen receptor alpha
Raloxifene
Estrogen-related receptor gamma
Estrogen-related receptor alpha
Cite
Citations (106)
Gene transfer studies have shown that estrogen regulation of specific genes is mediated by estrogen response elements (ERE). We report that binding of the estrogen receptor to the ERE can be detected by a gel retardation (band shift) assay. This binding interaction was highly sequence and receptor specific. Methylation interference analysis showed that the ERE contact sites of estrogen receptor displayed a perfect twofold rotational symmetry. This is compatible with estrogen receptor binding to the ERE as a head-to-head dimer.
Hormone response element
Estrogen receptor beta
Estrogen receptor alpha
Cite
Citations (199)
Cite
Citations (6)
Raloxifene
Estrogen receptor beta
Hormone response element
Estrogen receptor alpha
Cite
Citations (62)
Hormone response element
Estrogen receptor beta
PELP-1
Estrogen receptor alpha
Transcription
Cite
Citations (0)
Abstract A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERβ. By screening a rat prostate cDNA library, we detected ERβ as well as a novel isoform that we termed ERβ2. ERβ2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERβ hormone-binding domain. We also have evidence for the expression of both ERβ1 and ERβ2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERβ2 for 17β-estradiol (E2)[ dissociation constant (Kd ∼ 8 nm)] as compared with ERβ1 (Kd ∼ 1 nm). In vitro transcribed and translated ERβ1 and ERβ2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERβ1 and ERβ2 with each other as well as with ERα. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ERα and ERβ1, but not with ERβ2. In cotransfection experiments with expression plasmids for ERα, ERβ1, and ERβ2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERβ1 to E2 was similar to that of ERα although the maximal stimulation was approximately 50%. In contrast, ERβ2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERβ2 adds yet another facet to the possible cellular responses to estrogen.
Hormone response element
Estrogen receptor beta
Estrogen receptor alpha
Cite
Citations (131)