The Isoforms of Estrogen Receptor Alpha and Beta in Thyroid Cancer.
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ABSTRACT:
SUBMITTER: Gong Z
PROVIDER: S-EPMC9263191 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.
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Project description:PurposeTriple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERβ) as a possible endocrine therapy target in TNBC.MethodsThe expression and prognostic effect of ERβ isoforms were analyzed using TCGA breast tumor data, and the expression of ERβ isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERβ2 and ERβ5 were knocked down with siRNA, and ERβ2, ERβ5, and ERβ1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated.ResultsERβ2 and ERβ5 were the predominant endogenous forms of ERβ in TNBC tumors and cell lines. High ERβ2 predicted worse clinical outcome. Knockdown of endogenous ERβ2/ERβ5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERβ2/ERβ5 upregulation did the reverse, increasing survivin and these cell activities. ERβ1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERβ ligands.ConclusionsERβ2/ERβ5 and ERβ1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERβ isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.
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Project description:Estrogens have important effects on male and female social behavior. Despite growing knowledge of the anatomy and behavioral effects of the two predominant estrogen receptor subtypes in mammals (ERalpha and ERbeta), relatively little is known about how these receptors respond to salient environmental stimuli. Many seasonally breeding species respond to changing photoperiods that predict seasonal changes in resource availability. We characterized the effects of photoperiod on aggressive behavior in two species of Peromyscus that exhibit gonadal regression in short days. P. polionotus (old field mice) were more aggressive than P. maniculatus (deer mice) and both species were more aggressive in short days. We used immunocytochemistry and real-time polymerase chain reaction to characterize the effects of photoperiod on ERalpha and ERbeta expression. In both species ERalpha-immunoreactive staining in the posterior bed nucleus of the stria terminalis (BNST) was increased in short vs. long days. Both species had reduced ERbeta-immunoreactive expression in the posterior BNST in short days. In the medial amygdala ERbeta immunoreactivity was increased in long days for both species. Using real-time polymerase chain reaction on punch samples that included the BNST, we observed that ERalpha mRNA was increased and ERbeta mRNA was decreased in short days. These data suggest that the effects of photoperiod on ERalpha and ERbeta expression may thus have important behavioral consequences.
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Project description:Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.
| S-EPMC3639985 | biostudies-literature
Project description:Estrogen receptor beta (ER-beta) regulates diverse physiological functions in the human body. Current studies are confined to ER-beta1, and the functional roles of isoforms 2, 4, and 5 remain unclear. Full-length ER-beta4 and -beta5 isoforms were obtained from a prostate cell line, and they exhibit differential expression in a wide variety of human tissues/cell lines. Through molecular modeling, we established that only ER-beta1 has a full-length helix 11 and a helix 12 that assumes an agonist-directed position. In ER-beta2, the shortened C terminus results in a disoriented helix 12 and marked shrinkage in the coactivator binding cleft. ER-beta4 and -beta5 completely lack helix 12. We further demonstrated that ER-beta1 is the only fully functional isoform, whereas ER-beta2, -beta4, and -beta5 do not form homodimers and have no innate activities of their own. However, the isoforms can heterodimerize with ER-beta1 and enhance its transactivation in a ligand-dependent manner. ER-beta1 tends to form heterodimers with other isoforms under the stimulation of estrogens but not phytoestrogens. Collectively, these data support the premise that (i) ER-beta1 is the obligatory partner of an ER-beta dimer, whereas the other isoforms function as variable dimer partners with enhancer activity, and (ii) a single functional helix 12 in a dimer is sufficient for gene transactivation. Thus, ER-beta behaves like a noncanonical type-I receptor, and its action may depend on differential amounts of ER-beta1 homo- and heterodimers formed upon stimulation by a specific ligand. Our findings have provided previously unrecognized directions for studying ER-beta signaling and design of ER-beta-based therapies.
| S-EPMC1552044 | biostudies-literature
Project description:The estrogen receptor β (ERβ) functions as a tumor suppressor in glioblastoma (GBM) cells. However, the in vivo significance of endogenous ERβ and the roles of its isoforms in GBM are incompletely understood. Using ERβ isoform-specific PCR screening, we found that GBM cells predominantly express ERβ1 and ERβ5, along with low levels of ERβ2 and ERβ4. We observed greater ERβ5 expression in higher grades of glioma than in lower grades. In CRISPR-based ERβ knockout (KO) cells and ERβ KO cells uniquely expressing ERβ1 or ERβ5 only, ERβ1 significantly reduced proliferation. Compared with parental GBM cells, ERβ KO cells exhibited high migratory and invasive potentials, and reexpression of ERβ1 resulted in the reduction of this phenotype. Interestingly, ERβ5 expression increased foci formation and anchorage-independent growth of NIH3T3 cells and increased motile structure formation, including filopodia and ruffles in GBM cells. Only ERβ1-expressing tumors resulted in longer mouse survival. RNA-Seq analysis revealed unique pathways modulated by ERβ1 and ERβ5. Compared with ERβ KO cells, ERβ1 cells exhibited lower activation of mTOR signaling molecules, including p-mTOR, p-S6K, and p-S6, and ERβ5-expressing cells had enhanced mTOR downstream signaling. Unique proteins including several that function as regulators of mTOR, immunomodulatory, and apoptosis pathways bound to ERβ1 and ERβ5 isoforms. Our work confirms the tumor-suppressive potential of ERβ1 and reveals the acquired oncogenic ability of ERβ5 in GBM cells. ERβ isoform status and their unique interactions with oncogenic pathways may have important implications in GBM progression.Significance: These findings suggest that only ERβ isoform 1 has tumor suppressor function in GBM and that ERβ isoform switching contributes to GBM progression. Cancer Res; 78(12); 3176-89. ©2018 AACR.
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Project description:Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.
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Project description:Thyroid hormone (T(3)) activates nuclear receptor transcription factors, encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) genes, to regulate target gene expression. Several TR isoforms exist, and studies of null mice have identified some unique functions for individual TR variants, although considerable redundancy occurs, raising questions about the specificity of T(3) action. Thus, it is not known how diverse T(3) actions are regulated in target tissues that express multiple receptor variants. I have identified two novel TRbeta isoforms that are expressed widely and result from alternative mRNA splicing. TRbeta3 is a 44.6-kDa protein that contains an unique 23-amino-acid N terminus and acts as a functional receptor. TRDeltabeta3 is a 32.8-kDa protein that lacks a DNA binding domain but retains ligand binding activity and is a potent dominant-negative antagonist. The relative concentrations of beta3 and Deltabeta3 mRNAs vary between tissues and with changes in thyroid status, indicating that alternative splicing is tissue specific and T(3) regulated. These data provide novel insights into the mechanisms of T(3) action and define a new level of specificity that may regulate thyroid status in tissue.
| S-EPMC102140 | biostudies-literature
Project description:Metabolic modifications of tumor cells are hallmarks of cancer. They exhibit an altered metabolism that allows them to sustain higher proliferation rates in hostile environment outside the cell. In thyroid tumors, the expression of the estrogen-related receptor ? (ERR?), a major factor of metabolic adaptation, is closely related to the oxidative metabolism and the proliferative status of the cells. To elucidate the role played by ERR? in the glycolytic adaptation of tumor cells, we focused on the regulation of lactate dehydrogenases A and B (LDHA, LDHB) and the LDHA/LDHB ratio. Our study included tissue samples from 10 classical and 10 oncocytic variants of follicular thyroid tumors and 10 normal thyroid tissues, as well as samples from three human thyroid tumor cell lines: FTC-133, XTC.UC1 and RO82W-1. We identified multiple cis-acting promoter elements for ERR?, in both the LDHA and LDHB genes. The interaction between ERR? and LDH promoters was confirmed by chromatin immunoprecipitation assays and in vitro analysis for LDHB. Using knock-in and knock-out cellular models, we found an inverse correlation between ERR? expression and LDH activity. This suggests that thyroid tumor cells may reprogram their metabolic pathways through the up-regulation of ERR? by a process distinct from that proposed by the recently revisited Warburg hypothesis.
| S-EPMC3596295 | biostudies-literature
Project description:ObjectivesEndometriosis is a common gynecological disorder, characterized by the presence of endometrial-like tissue in the extrauterine location. The increasing estradiol concentration can influence endometriosis risk and estrogen receptor (ER) activity. Polymorphism in ER causes gene expression alteration and influences hormone-receptor interaction. This research aims to determine ER genetic polymorphisms in endometriosis pathogenesis.Materials and methodsThis study was performed on case-control polymorphisms, which compared 83 women with endometriosis and 76 women without endometriosis. However, the samples used for ER gene expression analysis and estrogen level measurement were obtained from 18 women with endometriosis and 18 women without endometriosis. Polymerase chain reaction-restriction fragment length polymorphism was used to determine ER genetic polymorphisms. Chi-square, Mann-Whitney test, Spearman's correlation (p), t-independent, and two-tailed tests were used to analyze the data.ResultsAssociation between the allele ERα rs9340799 A/G and endometriosis was significantly different (p=0.012), whereas rs2234693 T/C polymorphism showed no association with endometriosis. The correlation between the genotype frequencies of allele ERβ rs4986938 G/A and endometriosis was found significantly different (p=0.015; p=0.034).ConclusionEstradiol level and ERβ expression increases, polymorphism genotypes and alleles of ERβ rs4986938 G/A gene and allele frequency of ERα rs9340799 A/G gene have roles in endometriosis.
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