ER Ligand Binding Domain Research Articles

ESR1 mutations—a mechanism for acquired endocrine resistance in breast cancer.

Approximately 70% of breast cancers are oestrogen receptor α (ER) positive, and are, therefore, treated with endocrine therapies. However, about 25% of patients with primary disease and almost all patients with metastases will present with or eventually develop endocrine resistance. Despite the magnitude of this clinical challenge, the mechanisms underlying the development of resistance remain largely unknown. In the past 2 years, several studies unveiled gain-of-function mutations in ESR1, the gene encoding the ER, in approximately 20% of patients with metastatic ER-positive disease who received endocrine therapies, such as tamoxifen and aromatase inhibitors. These mutations are clustered in a 'hotspot' within the ligand-binding domain (LBD) of the ER and lead to ligand-independent ER activity that promotes tumour growth, partial resistance to endocrine therapy, and potentially enhanced metastatic capacity; thus, ER LBD mutations might account for a mechanism of acquired endocrine resistance in a substantial fraction of patients with metastatic disease. In general, the absence of detectable ESR1 mutations in patients with treatment-naive disease, and the correlation between the frequency of patients with tumours harbouring these mutations and the number of endocrine treatments received suggest that, under selective treatment pressure, clonal expansion of rare mutant clones occurs, leading to resistance. Preclinical and clinical development of rationale-based novel therapeutic strategies that inhibit these ER mutants has the potential to substantially improve treatment outcomes. We discuss the contribution of ESR1 mutations to the development of acquired resistance to endocrine therapy, and evaluate how mutated ER can be detected and targeted to overcome resistance and improve patient outcomes.

Abstract S3-05: Patient-derived xenograft study reveals endocrine therapy resistance of ER+ breast cancer caused by distinct ESR1 gene aberrations

Abstract Endocrine therapy resistance occurs in 50% of estrogen receptor positive (ER+) luminal breast cancers but the underlying mechanisms are poorly understood. To gain insight, we have taken advantage of whole-genome and RNA sequencing data of five late-stage hormone-resistant luminal breast tumors all of which have been successfully established as patient-derived mouse xenograft (PDX) models. Here we describe genetic alterations in ESR1-the gene coding for ERa-in three of these five tumors. They include an ESR1 point mutation (Y537S), a gene translocation causing an in-frame fusion between N-terminal ER and C-terminal Yes-associated protein 1 (YAP1), and ESR1 gene amplification. Functional characterization of the ESR1(Y537S) mutant and ESR1-YAP1 fusion in ER+ cell lines indicated that they both possess constitutive transcriptional activity and drive hormone-independent cell proliferation, mirroring the endocrine resistance of the originating tumors and the estradiol-independent growth of the PDX tumors. ESR1 (Y537S), a known gain-of-function experimental mutation in the ligand-binding domain of ER is not seen in primary breast cancer (TCGA data), suggesting it is a mutation associated with acquired resistance. Regarding the ESR1-YAP1 fusion gene, the truncated N-terminal ER fragment lacks the hormone-dependent transactivation domain (AF2) and the ligand binding domain but retains the hormone-independent transactivation domain (AF1) and therefore drives resistance to all endocrine approaches. As for ESR1 gene amplification, the tumor of origin, though resistant to aromatase inhibition, paradoxically responded to estradiol treatment, and this was recapitulated in the PDX model. Interestingly, acquired ESR1 gene amplification also occurred in long-term estrogen-deprived breast cancer MCF-7 cells which similarly regress upon estradiol exposure. Thus, ESR1 amplification may be a biomarker for paradoxical therapy with estradiol. Together, our focused study of advanced endocrine resistant luminal breast tumors revealed three distinct mutational mechanisms affecting the ESR1 gene that drive endocrine therapy resistance. Prevalence studies using RNAseq are underway to determine the frequency of somatic changes in the ESR1 gene in advanced breast cancer samples and additional ER+ PDX models. These additional data will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-05.

Exploration of Dimensions of Estrogen Potency: PARSING LIGAND BINDING AND COACTIVATOR BINDING AFFINITIES

The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity.

Development of an image analysis screen for estrogen receptor alpha (ERα) ligands through measurement of nuclear translocation dynamics

We have developed a robust high-content assay to screen for novel estrogen receptor alpha (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen utilizes a green fluorescent protein tagged-glucocorticoid/estrogen receptor (GFP-GRER) chimera which consisted of the N-terminus of the glucocorticoid receptor fused to the human ER ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands, and translocated to the nucleus in response to stimulation with ERα agonists or antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. The assay was validated with known ERα agonists and antagonists, and the Library of Pharmacologically Active Compounds (LOPAC 1280). Additionally, screening of crude natural product extracts demonstrated the robustness of the assay, and the ability to quantitate the effects of toxicity on nuclear translocation dynamics. The GFP-GRER nuclear translocation assay was very robust, with z′ values >0.7, CVs <5%, and has been validated with known ER ligands, and inclusion of cytotoxicity filters will facilitate screening of natural product extracts. This assay has been developed for future primary screening of synthetic, pure natural products, and natural product extracts libraries available at the National Cancer Institute at Frederick.

Evidence for Unique Mechanisms of Estrogen Receptor Degradation Elicited by Estradiol and ICI 182,780 (Fulvestrant).

Abstract Selective estrogen receptor down-regulators (SERDs), such as ICI 182780 (Fulvestrant), comprise a class of drugs that has been developed to inhibit estrogen receptor alpha (ER) activity in breast cancer. ICI is a pure antagonist that competitively binds to the ligand-binding domain of ER. The binding of ICI is thought to reduce the receptor's half-life by increasing protein turnover. However, the exact mechanism of ICI action, including whether or not receptor degradation is actually required for its antiestrogenic action, is currently unknown. Our laboratory has shown that a cytoplasmic ER mutant (ERΔNLS) lacking a nuclear localization sequence (NLS) is completely resistant to ICI-mediated degradation in both MCF-7 breast cancer cells and their ER-negative derivatives, C4-12 cells. Immunofluorescence and confocal microscopy were performed to assess localization of all ER proteins. These cells were also treated for different lengths of time with varying concentrations of both estradiol (E2) and ICI. Immunoblotting was performed to determine levels of ER protein. A competitive radioligand binding assay was also conducted to assess the ligand-binding capability of the ERΔNLS mutant receptor. Immunofluorescence and confocal imaging of stably transfected C4-12 cells has confirmed distinct subcellular localization of each ER protein. Wild-type ER localizes mainly to the nucleus, while ERΔNLS is cytoplasmic. As expected, treatment of either wild-type MCF-7 cells or C4-12 cells expressing wild-type ER with E2 or ICI caused degradation of ER protein. Treatment of MCF-7/ ERΔNLS or C4-12/ ERΔNLS cells with E2 caused degradation of ERΔNLS protein. In stark contrast, increasing concentrations of ICI failed to cause degradation of ERΔNLS. Furthermore, experiments performed in the presence of cycloheximide have shown that ICI does not modulate ERΔNLS half-life. The inability of ICI to degrade ERΔNLS is not due to an inability of ERΔNLS to bind ligand, since results from competitive radioligand binding assays show that the affinity of ERΔNLS for both E2 and ICI is equal to the affinity of wild-type ER for both ligands. We have recently cloned the SV40-NLS onto ERΔNLS, and confocal microscopy has confirmed that the SV40-NLS is sufficient to force ERΔNLS into the nucleus. I am currently using this construct to determine if it is the NLS domain itself or merely subcellular localization of ER that is important for ICI-mediated degradation. In conclusion, ER is a critical marker for response to antiestrogen therapy. Treatment with ICI is not without its shortcomings. A better understanding of the mechanism by which ICI degrades ER may lead to the development of better antiestrogen therapies to inhibit ER action with less toxicity and greater specificity. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4137.

Key elements for discriminating selective estrogen receptor modulators and selective receptor estrogen down regulators: a molecular approach to classify antiestrogens.

Abstract Abstract #3036 Breast cancer is the most common type of malignancy among women in the world. About 60% of breast tumours express the oestrogen receptor alpha (ERα) and are considered hormone-responsive. Thus endocrine therapies have been the treatment of choice. However, the estrogen-like agonist effect of the available selective estrogen receptor modulators such as tamoxifen and the development of resistances require the development of new treatments that act through different mechanisms.&amp;#x2028; The objective of our study is to design new anti-estrogens and tools that can help to understand the molecular mechanisms involved in ligand dependent modulation or degradation of ERα. It has been proposed that ligand dependent ERα degradation of antagonist may result from the presence of an aliphatic side chain on a steroidal body. Our aim is to exploit the structural characteristics of anti-estrogens to create new compounds with different lateral side chains for the study of the relationship between structure and activity of antiestrogens.&amp;#x2028; We selected the following compounds: RU 39411, RU 58668, which have similar structures of the steroid body, but different side-chain motifs: RU 39411 has a side chain similar to that of Tamoxifen, while RU 58668 has one similar to Faslodex. The third compound, EM 652, has a body of intermediate structure between stilbestrol and oestradiol while the side chain is similar to that of Raloxifen.&amp;#x2028; First, we analysed the degradation of ERα in the presence of the different ligands: Fulvestrant and RU 58668 induce degradation of ERα,in a protasome dependent manner, while RU 39411 and EM 652 have the same effect as Tamoxifen and stabilize ERα. To further analyze the molecular mechanism we generated three mutants of ERα; L540Q (in H12 of ligand binding domain), K362A (interaction with co-activators,) and A350V-D351Y (interaction with side chain of antiestrogens).&amp;#x2028; Using transient transfection of the different mutant constructs of MDA-MB231(ER-negative) cells, we tested the effect of the ligands on gene expression of a luciferase reporter gene under the control of an estrogen inducible promoter and ligand-induced degradation of ER. Our results demonstrate that antiestrogens can be classified into two groups based on structure and function: Tamoxifen and RU39411 fall into a first group, and Faslodex and RU58668 into a second group. However EM 652 has an intermediate behavior. Preliminary results on stable cell lines show that results from transient transfections can be reproduced.and confirm the importance of the integrity of H12 for antagonist activity. Molecular modelisation demonstrates that variations in the structure of the ER ligand binding domain in the presence of different antiestrogens correlate with functional differences.&amp;#x2028; We have used molecular studies to screen and classify antiestrogen compounds into SERM or SERD with the aim to develop novel molecules for endocrine treatment of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3036.

C/EBPα:AP-1 Leucine Zipper Heterodimers Bind Novel DNA Elements, Activate the PU.1 Promoter, and Direct Monocyte Lineage Commitment More Potently Than C/EBPα Homodimers or AP-1.

The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA-binding. Members of the C/EBP, AP-1, and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind specific DNA motifs. Here we demonstrate that C/EBPα also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPα zippers with c-Jun, JunB, or c-Fos, but not with c-Maf or MafB. In addition, C/EBPα co-ips c-Jun, JunB, or c-Fos, either expressed in 293T cells or as endogenous proteins in a myeloid cell line. Affinity of C/EBPα for AP-1 proteins appears weaker in this assay than affinity of C/EBPα for the related C/EBPβ, suggesting that interaction of C/EBP and AP-1 proteins is not primarily cross-inhibitory but instead generates heterodimers capable of binding novel DNA elements and inducing unique biological effects. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. This approach is advantageous over efforts to fuse bZIP proteins together as zippering of tethered proteins may be sterically hindered and disruptive to the remainder of the protein. We find that C/EBPαLZE:C/EBPαLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site, and C/EBPαLZE:c-JunLZK a hybrid element identified as 5′-TTGCGTCAT by oligonucleotide selection. In contrast, c-Jun homodimers selected the consensus site 5′-TGACGTCAT, and C/EBPα homodimers selected 5′-NNGNNGCAAC. Note that 5′-TTGC in the selected hybrid site corresponds to 5′-GCAA in the C/EBPα site. In lineage-negative murine myeloid progenitors, either C/EBPα-ER or c-Jun-ER potently induce monocyte as compared to granulocyte development. Using LZE:LZK proteins linked to the ER ligand-binding domain, we find that C/EBPα:c-Jun or C/EBPα:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPα or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. We propose that C/EBPα induction of monopoiesis requires zippering with endogenous c-Jun and that conversely induction of monopoiesis by c-Jun requires zippering with endogenous C/EBPα. Finally, using Ba/F3 lines stably expressing LZE:LZK ER fusion proteins, we find that C/EBPα:c-Jun binds the endogenous PU.1 promoter as assessed by chromatin immunoprecipitation and that exposure of cells expressing C/EBPαLZK-ER and c-JunLZE-ER to estradiol induces PU.1 mRNA expression as assessed by quantitative RT-PCR, whereas C/EBPαLZK-ER in combination with C/EBPαLZE-ER did not induce PU.1 mRNA in Ba/F3 cells. In addition, both endogenous C/EBPα and c-Jun were detected by the ChIP assay in the vicinity of the PU.1 promoter in 32Dcl3 myeloid cells. Thus, induction of PU.1 may contribute to stimulation of monopoiesis by C/EBPα:c-Jun heterodimers. In addition, as C/EBP and AP-1 proteins are widely expressed, zippering between these bZIP subfamilies may have broad biologic relevance. To gain insight into the spectrum of genes activated specifically by this complex, we have initiated microarray studies using our Ba/F3 cell lines.

Structural Basis for an Unexpected Mode of SERM-Mediated ER Antagonism

Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.

CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor

AbstractPotential redundancy among members of the CCAAT/enhancer-binding protein (C/EBP) family in myeloid cells is indicated by the ability of C/EBPβ to replace C/EBPα in vivo, by the expression of granulocyte colony-stimulating factor receptor (G-CSFR) on C/EBPα−/− cell lines, and by our finding that as with C/EBPα–estrogen receptor (C/EBPα-ER), either C/EBPβ-ER or C/EBPδ-ER can induce terminal granulopoiesis in 32D cl3 cells. To assess the consequences of globally inhibiting C/EBPs, we employed KαER, containing a Kruppel-associated box (KRAB) transrepression domain, the C/EBPα DNA-binding domain, and an ER ligand-binding domain. C/EBPs have a common DNA-binding consensus, and activation of KαER repressed transactivation by endogenous C/EBPs 50-fold and reduced endogenous G-CSFR expression. In 32D cl3 cells coexpressing exogenous G-CSFR, activation of KαER prevented and even reversed myeloperoxidase, lysozyme, lactoferrin, and C/EBPε RNA induction by G-CSF. In contrast, induction of PU.1 and CD11b, a gene regulated by PU.1 but not by C/EBPs, was unaffected. A KαER variant incapable of binding DNA owing to an altered leucine zipper did not affect 32D cl3 differentiation. Transduction of KαER into murine hematopoietic progenitor cells suppressed the formation of granulocyte colony-forming units, even in cytokines that enable C/EBPα−/−progenitors to differentiate into neutrophils. The formation of macrophage and of granulocyte-macrophage colony-forming units were also inhibited, but erythroid burst-forming units grew normally. Thus, in 32D cl3 cells and perhaps normal progenitors, C/EBPs are required for granulopoiesis beyond their ability to induce receptors for G-CSF and other cytokines. One requisite activity may be activation of the C/EBPε gene by C/EBPα, as either C/EBPα-ER or C/EBPβ-ER rapidly elevated C/EBPε RNA in 32D cl3 cells in the presence of cycloheximide but not actinomycin D.

Different Classes of Coactivators Recognize Distinct but Overlapping Binding Sites on the Estrogen Receptor Ligand Binding Domain

We have analyzed interaction of coactivators with the wild-type estrogen receptor alpha (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Molecular basis of agonism and antagonism in the oestrogen receptor.

Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Regulation of Cre Recombinase Activity by Mutated Estrogen Receptor Ligand-Binding Domains

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17β-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.

Aberrant oestrogen receptor species in human meningioma tissue

Meningiomas are very rich in progestin receptors (PR) whereas oestrogen receptors (ER) are seldomly found and only at low concentrations. These tumours might possess an ER which is defective in oestrogen binding but still functional in stimulating oestrogen-responsive genes such as PR. In human meningiomas a polymerase chain reaction fragment including the DNA binding domain, the hinge region and the ligand binding domain of ER was amplified. The size of the fragment obtained was as expected from wild type mRNA sequences. Moreover, a variant, which was overexpressed in meningiomas, with a major deletion in exons 2–6 was detected.