RESUMO
Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.
Assuntos
Benzamidas , Cromatina , Fenantrenos , Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/genética , Mifepristona/farmacologia , Complexo Mediador/metabolismo , Glucocorticoides/farmacologia , Glucocorticoides/metabolismo , Dexametasona/farmacologiaRESUMO
A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.
Assuntos
Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Mutação de Sentido Incorreto , Coativador 1 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/genética , Sítios de Ligação/genética , Resistência a Medicamentos/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Cinética , Ligantes , Simulação de Dinâmica Molecular , Coativador 1 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.
Assuntos
Processamento Alternativo , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/metabolismo , Análise Serial de Tecidos , Transcrição GênicaRESUMO
OBJECTIVE: The nuclear receptor PPARγ is the master regulator of adipocyte differentiation, distribution, and function. In addition, PPARγ induces terminal differentiation of several epithelial cell lineages, including colon epithelia. Loss-of-function mutations in PPARG result in familial partial lipodystrophy subtype 3 (FPDL3), a rare condition characterized by aberrant adipose tissue distribution and severe metabolic complications, including diabetes. Mutations in PPARG have also been reported in sporadic colorectal cancers, but the significance of these mutations is unclear. Studying these natural PPARG mutations provides valuable insights into structure-function relationships in the PPARγ protein. We functionally characterized a novel FPLD3-associated PPARγ L451P mutation in helix 9 of the ligand binding domain (LBD). Interestingly, substitution of the adjacent amino acid K450 was previously reported in a human colon carcinoma cell line. METHODS: We performed a detailed side-by-side functional comparison of these two PPARγ mutants. RESULTS: PPARγ L451P shows multiple intermolecular defects, including impaired cofactor binding and reduced RXRα heterodimerisation and subsequent DNA binding, but not in DBD-LBD interdomain communication. The K450Q mutant displays none of these functional defects. Other colon cancer-associated PPARγ mutants displayed diverse phenotypes, ranging from complete loss of activity to wildtype activity. CONCLUSIONS: Amino acid changes in helix 9 can differently affect LBD integrity and function. In addition, FPLD3-associated PPARγ mutations consistently cause intra- and/or intermolecular defects; colon cancer-associated PPARγ mutations on the other hand may play a role in colon cancer onset and progression, but this is not due to their effects on the most well-studied functional characteristics of PPARγ.
Assuntos
Lipodistrofia Parcial Familiar/genética , Mutação de Sentido Incorreto , PPAR gama/genética , Adulto , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Células HEK293 , Humanos , Lipodistrofia Parcial Familiar/patologia , PPAR gama/química , PPAR gama/metabolismo , Fenótipo , Multimerização ProteicaRESUMO
Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antagonistas do Receptor de Estrogênio/administração & dosagem , Receptor alfa de Estrogênio/antagonistas & inibidores , Indazóis/administração & dosagem , Mutação , Administração Oral , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Indazóis/química , Indazóis/farmacologia , Células MCF-7 , Camundongos , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17ß-estradiol (E2) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E2-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E2-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E2, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.
Assuntos
Estrogênios/farmacologia , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Tretinoína/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Luciferases/metabolismo , Células MCF-7 , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: To date, all studies conducted on breast cancer diagnosis have focused on the expression of the full-length 66-kDa estrogen receptor alpha (ERα66). However, much less attention has been paid to a shorter 46-kDa isoform (ERα46), devoid of the N-terminal region containing the transactivation function AF-1. Here, we investigated the expression levels of ERα46 in breast tumors in relation to tumor grade and size, and examined the mechanism of its generation and its specificities of coregulatory binding and its functional activities. METHODS: Using approaches combining immunohistochemistry, Western blotting, and proteomics, antibodies allowing ERα46 detection were identified and the expression levels of ERα46 were quantified in 116 ERα-positive human breast tumors. ERα46 expression upon cellular stress was studied, and coregulator bindings, transcriptional, and proliferative response were determined to both ERα isoforms. RESULTS: ERα46 was expressed in over 70% of breast tumors at variable levels which sometimes were more abundant than ERα66, especially in differentiated, lower-grade, and smaller-sized tumors. We also found that ERα46 can be generated via internal ribosome entry site-mediated translation in the context of endoplasmic reticulum stress. The binding affinities of both unliganded and fully-activated receptors towards co-regulator peptides revealed that the respective potencies of ERα46 and ERα66 differ significantly, contributing to the differential transcriptional activity of target genes to 17ß estradiol (E2). Finally, increasing amounts of ERα46 decrease the proliferation rate of MCF7 tumor cells in response to E2. CONCLUSIONS: We found that, besides the full-length ERα66, the overlooked ERα46 isoform is also expressed in a majority of breast tumors. This finding highlights the importance of the choice of antibodies used for the diagnosis of breast cancer, which are able or not to detect the ERα46 isoform. In addition, since the function of both ERα isoforms differs, this work underlines the need to develop new technologies in order to discriminate ERα66 and ERα46 expression in breast cancer diagnosis which could have potential clinical relevance.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Processamento Alternativo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Estresse do Retículo Endoplasmático , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Ligação Proteica , Biossíntese de Proteínas , Isoformas de Proteínas , Proteoma , Proteômica/métodos , Estudos RetrospectivosRESUMO
BACKGROUND: NR2E1 (Tlx) is an orphan nuclear receptor that regulates the maintenance and self-renewal of neural stem cells, and promotes tumourigenesis. Nr2e1-null mice exhibit reduced cortical and limbic structures and pronounced retinal dystrophy. NR2E1 functions mainly as a repressor of gene transcription in association with the co-repressors atrophin-1, LSD1, HDAC and BCL11A. Recent evidence suggests that NR2E1 also acts as an activator of gene transcription. However, co-activator complexes that interact with NR2E1 have not yet been identified. In order to find potential novel co-regulators for NR2E1, we used a microarray assay for real-time analysis of co-regulator-nuclear receptor interaction (MARCoNI) that contains peptides representing interaction motifs from potential co-regulatory proteins, including known co-activator nuclear receptor box sequences (LxxLL motif). RESULTS: We found that NR2E1 binds strongly to an atrophin-1 peptide (Atro box) used as positive control and to 19 other peptides that constitute candidate NR2E1 partners. Two of these proteins, p300 and androgen receptor (AR), were further validated by reciprocal pull-down assays. The specificity of NR2E1 binding to peptides in the array was evaluated using two single amino acid variants, R274G and R276Q, which disrupted the majority of the binding interactions observed with wild-type NR2E1. The decreased binding affinity of these variants to co-regulators was further validated by pull-down assays using atrophin1 as bait. Despite the high conservation of arginine 274 in vertebrates, its reduced interactions with co-regulators were not significant in vivo as determined by retinal phenotype analysis in single-copy Nr2e1-null mice carrying the variant R274G. CONCLUSIONS: We showed that MARCoNI is a specific assay to test interactions of NR2E1 with candidate co-regulators. In this way, we unveiled 19 potential co-regulator partners for NR2E1, including eight co-activators. All the candidates here identified need to be further validated using in vitro and in vivo models. This assay was sensitive to point mutations in NR2E1 ligand binding domain making it useful to identify mutations and/or small molecules that alter binding of NR2E1 to protein partners.
Assuntos
Ligantes , Receptores Nucleares Órfãos/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Sequência de Aminoácidos , Animais , Proteínas de Transporte , Descoberta de Drogas , Variação Genética , Humanos , Camundongos , Camundongos Knockout , Receptores Nucleares Órfãos/química , Receptores Nucleares Órfãos/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.
Assuntos
Proteínas de Transporte/genética , Proteínas Nucleares/genética , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias da Próstata/genética , Receptores Androgênicos/genética , Proteínas Repressoras/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Transdução de SinaisRESUMO
Treatment-induced mutations in the ligand-binding domain of the androgen receptor (AR) are known to change antagonists into agonists. Recently, the F877L mutation has been described to convert enzalutamide into an agonist. This mutation was seen to co-occur in the endogenous AR allele of LNCaP cells, next to the T878A mutation. Here, we studied the effects of enzalutamide on the F877L and T878A mutants, as well as the double-mutant AR (F877L/T878A). Molecular modeling revealed favorable structural changes in the double-mutant AR that lead to a decrease in steric clashes for enzalutamide. Ligand-binding assays confirmed that the F877L mutation leads to an increase in relative binding affinity for enzalutamide, but only the combination with the T878A mutation resulted in a strong agonistic activity. This correlated with changes in coregulator recruitment and chromatin interactions. Our data show that enzalutamide is only a very weak partial agonist of the AR F877L, and a strong partial agonist of the double-mutant AR. Mol Cancer Ther; 15(7); 1702-12. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Códon , Resistencia a Medicamentos Antineoplásicos/genética , Mutação , Feniltioidantoína/análogos & derivados , Receptores Androgênicos/genética , Substituição de Aminoácidos , Antagonistas de Androgênios/farmacologia , Antineoplásicos/química , Benzamidas , Linhagem Celular Tumoral , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Nitrilas , Feniltioidantoína/química , Feniltioidantoína/farmacologia , Ligação Proteica , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
The aim of the present study was to investigate modulation of the interaction of ERα and ERß with coregulators in the ligand dependent responses induced by the ER antagonistic compounds 4OHT and fulvestrant. Comparison with the modulation index (MI) profiles for the ER agonist estradiol (E2) will elucidate whether differences in the (ant)agonist dependent interaction of ERα and ERß with coregulators expressed in MI profiles contribute to the differences in (ant)agonist responses. To this end, the selected ER antagonistic compounds were first characterized for intrinsic relative potency and efficacy towards ERα and ERß using ER selective U2OS reporter gene assays, and subsequently tested for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay. Results obtained indicate a preference of 4OHT to antagonize ERß and find fulvestrant to be less ER specific. MARCoNI assay responses reveal that ERα and ERß mediated interaction with coregulators expressed in MI profiles are similar for 4OHT and fulvestrant and generally opposite to the MI profile of the ER agonist E2. Hierarchical clustering based on the MI profiles appeared able to clearly discriminate the two compounds with ER antagonistic properties from the ER agonist E2. Taken together the data reveal that modulation of the interaction of ERs with coregulators discriminates ER agonists from antagonists but does not discriminate between the less specific ER antagonist fulvestrant and the preferential ERß antagonistic compound 4OHT. It is concluded that differences in modulation of the interaction of ERα and ERß with coregulators contribute to the differences in ligand dependent responses induced by ER agonists and ER antagonists but the importance of the subtle differences in modulation of the interaction of ERs with coregulators between the ER antagonistic compounds 4OHT and fulvestrant for the ultimate biological effect remains to be established.
Assuntos
Estradiol/análogos & derivados , Tamoxifeno/análogos & derivados , Linhagem Celular , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Fulvestranto , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Análise em Microsséries , Ligação Proteica/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Tamoxifeno/farmacologiaRESUMO
The aim of the present study was to investigate modulation of the interaction of the ERα and ERß with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERα and ERß agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERα and ERß mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERß cells with variable ERα/ERß ratio, and finally for ligand dependent modulation of the interaction of ERα and ERß with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERß ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERα agonists activate cell proliferation whereas ERß suppresses ERα mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERα or ERß activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERα or ERß activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays. It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERα and ERß and the cellular ERα/ERß ratio whereas differences in the modulation of the interaction of the ERα and ERß with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Células Tumorais CultivadasRESUMO
To further develop an integrated in vitro testing strategy for replacement of in vivo tests for (anti-)estrogenicity testing, the ligand-modulated interaction of coregulators with estrogen receptor α was assessed using a PamChip® plate. The relative estrogenic potencies determined, based on ERα binding to coregulator peptides in the presence of ligands on the PamChip® plate, were compared to the relative estrogenic potencies as determined in the in vivo uterotrophic assay. The results show that the estrogenic potencies predicted by the 57 coactivators on the peptide microarray for 18 compounds that display a clear E2 dose-dependent response (goodness of fit of a logistic dose-response model of 0.90 or higher) correlated very well with their in vivo potencies in the uterotrophic assay, i.e., coefficient of determination values for 30 coactivators higher than or equal to 0.85. Moreover, this coregulator binding assay is able to distinguish ER agonists from ER antagonists: profiles of selective estrogen receptor modulators, such as tamoxifen, were distinct from those of pure ER agonists, such as dienestrol. Combination of this coregulator binding assay with other types of in vitro assays, e.g., reporter gene assays and the H295R steroidogenesis assay, will frame an in vitro test panel for screening and prioritization of chemicals, thereby contributing to the reduction and ultimately the replacement of animal testing for (anti-)estrogenic effects.
Assuntos
Disruptores Endócrinos/farmacologia , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Procedimentos Analíticos em Microchip/métodos , Análise Serial de Proteínas/métodos , Ligação Proteica , Disruptores Endócrinos/química , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Estrogênios/química , Humanos , Reprodutibilidade dos TestesRESUMO
With current techniques, it remains a challenge to assess coregulator binding of nuclear receptors, for example, the estrogen receptor alpha (ERα). ERα is critical in many breast tumors and is inhibited by antiestrogens such as tamoxifen in cancer therapy. ERα is also modified by acetylation and phosphorylation that affect responses to the antiestrogens as well as interactions with coregulators. Phosphorylation of ERα at Ser305 is one of the mechanisms causing tamoxifen resistance. Detection of resistance in patient samples would greatly facilitate clinical decisions on treatment, in which such patients would receive other treatments such as aromatase inhibitors or fulvestrant. Here we describe a coregulator peptide array that can be used for high-throughput analysis of full-length estrogen receptor binding. The peptide chip can detect ERα binding in cell and tumor lysates. We show that ERα phosphorylated at Ser305 associates stronger to various coregulator peptides on the chip. This implies that ERαSer305 phosphorylation increases estrogen receptor function. As this is also detected in a breast tumor sample of a tamoxifen-insensitive patient, the peptide array, as described here, may be applicable to detect tamoxifen resistance in breast tumor samples at an early stage of disease and contribute to personalized medicine.