Nuclear Receptor Corepressor Research Articles

Akt-Induced Phosphorylation of N-CoR at Serine 1450 Contributes to Its Misfolded Conformational Dependent Loss (MCDL) in Acute Myeloid Leukemia of the M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic co-repressor complex that plays an important role in the control of cellular growth and differentiation. As shown by us recently, the growth suppressive function of N-CoR largely relies on its capacity to repress Flt3, a key regulator of cellular gorwth during normal and malignant hematopoesis. We further demonstrated how de-repression of Flt3 due to the misfolded conformation dependent loss (MCDL) of N-CoR contributed to malignant growth in acute myeloid leukemia (AML). However, the molecular mechanism underlying the MCDL of N-CoR and its implication in AML pathogenesis is not fully understood. Here, we report that Akt-induced phosphorylation of N-CoR at the consensus Akt motif is crucial for its misfolding and subsequent loss in AML (AML-M5). N-CoR displayed significantly higher level of serine specific phosphorylation in almost all AML-M5 derived cells and was subjected to processing by AML-M5 specific aberrant protease activity. To identify the kinase linked to N-CoR phosphorylation, a library of activated kinases was screened with the extracts of AML cells; leading to the identification of Akt as the putative kinase linked to N-CoR phosphorylation. Consistent with this finding, a constitutively active Akt consistently phosphorylated N-CoR leading to its misfolding; while the therapeutic and genetic ablation of Akt largely abrogated the MCDL of N-CoR in AML-M5 cells. Site directed mutagenic analysis of N-CoR identified serine 1450 as the crucial residue whose phosphorylation by Akt was essential for the misfolding and loss of N-CoR protein. Moreover, Akt-induced phosphorylation of N-CoR contributed to the de-repression of Flt3, suggesting a cross talk between Akt signaling and N-CoR misfolding pathway in the pathogenesis of AML-M5. The N-CoR misfolding pathway could be the common downstream thread of pleiotropic Akt signaling activated by various oncogenic insults in some subtypes of leukemia and solid tumors.

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Toward Position-independent Retroviral Vector Expression in Pluripotent Stem Cells

Anti-androgen effects of the pyrethroid pesticide cypermethrin on interactions of androgen receptor with corepressors

To clarify whether the mechanism of androgen receptor (AR) antagonism of the pyrethroid pesticide cypermethrin associates with the interactions between the AR and corepressors silencing mediator for thyroid hormone receptors (SMRT) and nuclear receptor corepressor (NCoR), we have developed the mammalian two-hybrid assays. The AR N-terminal domain 1–660 amino acid residues were subcloned into the plasmid pVP16 to construct VP16-ARNTD. The C-terminal receptor interaction domains (RIDs) of SMRT and NCoR were used to construct pM-SMRT and pM-NCoR. The constructed vectors pVP16-ARNTD, pM-SMRT or pM-NCoR, the reporter pG5CAT and the control pCMVβ were cotranfected into the CV-1 cells. The cells were treated with cypermethrin at the indicated concentrations. The AR N terminus interacted with RIDs of SMRT and NCoR. The interactions between the AR and corepressors SMRT and NCoR were enhanced by cypermethrin, and the significant enhancement was detected at the concentration of 10−5M. The mammalian two-hybrid assays demonstrate the utility to detect the interactions of the AR with SMRT and NCoR. Cypermethrin functions as an anti-androgen by enhancing the associations of the AR with SMRT and NCoR. We provide a novel mechanism in anti-androgen action of cypermethrin associated with the recruitment of SMRT and NCoR to AR.

Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR

Rett syndrome (RTT) is an X-linked human neurodevelopmental disorder with features of autism and severe neurological dysfunction in females. RTT is caused by mutations in methyl-CpG-binding protein 2 (MeCP2), a nuclear protein that, in neurons, regulates transcription, is expressed at high levels similar to that of histones, and binds to methylated cytosines broadly across the genome. By phosphotryptic mapping, we identify three sites (S86, S274 and T308) of activity-dependent MeCP2 phosphorylation. Phosphorylation of these sites is differentially induced by neuronal activity, brain-derived neurotrophic factor, or agents that elevate the intracellular level of 3',5'-cyclic AMP (cAMP), indicating that MeCP2 may function as an epigenetic regulator of gene expression that integrates diverse signals from the environment. Here we show that the phosphorylation of T308 blocks the interaction of the repressor domain of MeCP2 with the nuclear receptor co-repressor (NCoR) complex and suppresses the ability of MeCP2 to repress transcription. In knock-in mice bearing the common human RTT missense mutation R306C, neuronal activity fails to induce MeCP2 T308 phosphorylation, suggesting that the loss of T308 phosphorylation might contribute to RTT. Consistent with this possibility, the mutation of MeCP2 T308A in mice leads to a decrease in the induction of a subset of activity-regulated genes and to RTT-like symptoms. These findings indicate that the activity-dependent phosphorylation of MeCP2 at T308 regulates the interaction of MeCP2 with the NCoR complex, and that RTT in humans may be due, in part, to the loss of activity-dependent MeCP2 T308 phosphorylation and a disruption of the phosphorylation-regulated interaction of MeCP2 with the NCoR complex.

Oncogenic Actions of the Nuclear Receptor Corepressor (NCOR1) in a Mouse Model of Thyroid Cancer

Studies have suggested that the nuclear receptor corepressor 1 (NCOR1) could play an important role in human cancers. However, the detailed molecular mechanisms by which it functions in vivo to affect cancer progression are not clear. The present study elucidated the in vivo actions of NCOR1 in carcinogenesis using a mouse model (ThrbPV/PV mice) that spontaneously develops thyroid cancer. ThrbPV/PV mice harbor a dominantly negative thyroid hormone receptor β (TRβ) mutant (denoted as PV). We adopted the loss-of-the function approach by crossing ThrbPV mice with mice that globally express an NCOR1 mutant protein (NCOR1ΔID) in which the receptor interaction domains have been modified so that it cannot interact with the TRβ, or PV, in mice. Remarkably, expression of NCOR1ΔID protein reduced thyroid tumor growth, markedly delayed tumor progression, and prolonged survival of ThrbPV/PVNcor1 ΔID/ΔID mice. Tumor cell proliferation was inhibited by increased expression of cyclin-dependent kinase inhibitor 1 (p21waf1/cip1; Cdkn1A), and apoptosis was activated by elevated expression of pro-apoptotic BCL-Associated X (Bax). Further analyses showed that p53 was recruited to the p53-binding site on the proximal promoter of the Cdkn1A and the Bax gene as a co-repressor complex with PV/NCOR1/histone deacetylas-3 (HDAC-3), leading to repression of the Cdkn1A as well as the Bax gene in thyroids of ThrbPV/PV mice. In thyroids of ThrbPV/PVNcor1 ΔID/ΔID mice, the p53/PV complex could not recruit NCOR1ΔID and HDAC-3, leading to de-repression of both genes to inhibit cancer progression. The present studies provided direct evidence in vivo that NCOR1 could function as an oncogene via transcription regulation in a mouse model of thyroid cancer.

Reciprocal Negative Cross-Talk between Liver X Receptors (LXRs) and STAT1: Effects on IFN-γ–Induced Inflammatory Responses and LXR-Dependent Gene Expression

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ-induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ-induced genes in primary microglia and in a model of IFN-γ-induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ-mediated immune responses and in the regulation of lipid metabolism.

Lipin1 regulates PPARγ transcriptional activity

PPARγ (peroxisome-proliferator-activated receptor γ) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPARγ is largely unknown. The results of the present study show that lipin1 activates PPARγ by releasing co-repressors, NCoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPARγ in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPARγ, but not PPARα. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1α (PPAR co-activator 1α). The physical interaction between lipin1 and PPARγ occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPARγ. The action of lipin1 as a co-activator of PPARγ enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPARγ activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPARα activation.

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

Rev-Erbα and Rev-Erbβ are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm1,2, metabolism3,4, and inflammatory responses5. Rev-Erbs function as transcriptional repressors by recruiting NCoR/HDAC3 co-repressor complexes to Rev-Erb response elements in enhancers and promoters of target genes6-8, but the molecular basis for cell-specific programs of repression is not known. Here, we present evidence that in macrophages, Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby mRNAs, implying a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a method that quantifies nascent 5′ ends (5′-GRO-Seq), we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for direct roles of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG–binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex– and the NuRD complex–associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

Nuclear receptor corepressor (NCOR1) regulates in vivo actions of a mutated thyroid hormone receptor α

Genetic evidence from patients with mutations of the thyroid hormone receptor α gene (THRA) indicates that the dominant negative activity of mutants underlies the pathological manifestations. However, the molecular mechanisms by which TRα1 mutants exert dominant negative activity in vivo are not clear. We tested the hypothesis that the severe hypothyroidism in patients with THRA mutations is due to an inability of TRα1 mutants to properly release the nuclear corepressors (NCORs), thereby inhibiting thyroid hormone-mediated transcription activity. We crossed Thra1(PV) mice, expressing a dominant negative TRα1 mutant (TRα1PV), with mice expressing a mutant Ncor1 allele (Ncor1(ΔID) mice) that cannot recruit the TR or PV mutant. TRα1PV shares the same C-terminal mutated sequences as those of patients with frameshift mutations of the THRA gene. Remarkably, NCOR1ΔID ameliorated abnormalities in the thyroid-pituitary axis of Thra1(PV/+) mice. The severe retarded growth, infertility, and delayed bone development were partially reverted in Thra1(PV/+) mice expressing NCOR1ΔID. The impaired adipogenesis was partially corrected by de-repression of peroxisome-proliferator activated receptor γ and CCAAT/enhancer-binding protein α gene, due to the inability of TRα1PV to recruit NCOR1ΔID to form a repressor complex. Thus, the aberrant recruitment of NCOR1 by TRα1 mutants could lead to clinical hypothyroidism in humans. Therefore, therapies aimed at the TRα1-NCOR1 interaction or its downstream actions could be tested as potential targets in treating TRα1 mutant-mediated hypothyroidism in patients.

Emerging roles of the corepressors NCoR1 and SMRT in homeostasis

Epigenetic regulation of gene expression is strongly influenced by the accessibility of nucleosomal DNA or the state of chromatin compaction. In this context, coregulators, including both coactivators and corepressors, are pivotal intermediates that bridge chromatin-modifying enzymes and transcription factors. NCoR1 (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) are among the best-characterized corepressors from a molecular point of view. These coregulators have conserved orthologs in lower organisms, which underscores their functional importance. Here we summarize the results from recent in vivo studies that reveal the wide-ranging roles of NCoR1 and SMRT in developmental as well as homeostatic processes, including metabolism, inflammation, and circadian rhythms. We also discuss the potential implications of NCoR1 and SMRT regulation of pathways ranging from genomic stability and carcinogenesis to metabolic diseases such as type 2 diabetes.

A New Strategy for Selective Targeting of Progesterone Receptor With Passive Antagonists

Currently available progesterone (P4) receptor (PR) antagonists, such as mifepristone (RU486), lack specificity and display partial agonist properties, leading to potential drawbacks in their clinical use. Recent x-ray crystallographic studies have identified key contacts involved in the binding of agonists and antagonists with PR opening the way for a new rational strategy for inactivating PR. We report here the synthesis and characterization of a novel class of PR antagonists (APRn) designed from such studies. The lead molecule, the homosteroid APR19, displays in vivo endometrial anti-P4 activity. APR19 inhibits P4-induced PR recruitment and transactivation from synthetic and endogenous gene promoters. Importantly, it exhibits high PR selectivity with respect to other steroid hormone receptors and is devoid of any partial agonist activity on PR target gene transcription. Two-hybrid and immunostaining experiments reveal that APR19-bound PR is unable to interact with either steroid receptor coactivators 1 and 2 (SRC1 and SCR2) or nuclear receptor corepressor (NcoR) and silencing mediator of retinoid acid and thyroid hormone receptor (SMRT), in contrast to RU486-PR complexes. APR19 also inhibits agonist-induced phosphorylation of serine 294 regulating PR transcriptional activity and turnover kinetics. In silico docking studies based on the crystal structure of the PR ligand-binding domain show that, in contrast to P4, APR19 does not establish stabilizing hydrogen bonds with the ligand-binding cavity, resulting in an unstable ligand-receptor complex. Altogether, these properties highly distinguish APR19 from RU486 and likely its derivatives, suggesting that it belongs to a new class of pure antiprogestins that inactivate PR by a passive mechanism. These specific PR antagonists open new perspectives for long-term hormonal therapy.

Interaction of silencing mediator for retinoid and thyroid receptors with steroid and xenobiotic receptor on multidrug resistance 1 promoter

AimsThe steroid and xenobiotic receptor (SXR) regulates the transcription of its target genes by interacting with various nuclear receptor cofactors. We have previously shown that silencing mediator for retinoid and thyroid receptors (SMRT) interacts with SXR even in the presence of rifampicin on cytochrome P450 monooxygenase 3A4 (CYP3A4) promoter in HepG2 cells. To examine the specificity of such interaction, the involvement of SMRT on SXR-mediated transcription through multidrug resistance (MDR) 1 gene promoter was examined using LS174T intestine-derived clonal cells. Main methodsTransient transfection-based reporter gene assay was carried out to examine the effect of SMRT or nuclear receptor corepressor (NCoR) on SXR-mediated transcription in LS174T cells. Semi-quantitative RT-PCR was performed to confirm the expression of MDR1 mRNA in LS174T cells. To examine the interaction of SMRT with SXR, we carried out mammalian one-hybrid assay in CV-1 cells and immunoprecipitation study in HEK-293 cells. Key findingsSMRT, but not NCoR suppressed rifampicin-induced SXR-mediated transcription. The SXR-mediated MDR1 mRNA expression was augmented in the presence of rifampicin, whereas it suppressed the expression following the overexpression of SMRT. In mammalian one-hybrid assay, only SMRT but not NCoR interacted with SXR on MDR1 promoter in the presence of rifampicin. In immunoprecipitation study, SMRT bound to SXR regardless of the presence or absence of rifampicin. SignificanceSMRT may be recruited in the SXR-cofactor complex even in the presence of ligand. SMRT may be involved not only in SXR-mediated suppression without ligand, but also in ligand-activated transcription to suppress the overactivation of transcription.

Human IL10 Gene Repression by Rev-erbα Ameliorates Mycobacterium tuberculosis Clearance

Nuclear receptors modulate macrophage effector functions, which are imperative for clearance or survival of mycobacterial infection. The adopted orphan nuclear receptor Rev-erbα is a constitutive transcriptional repressor as it lacks AF2 domain and was earlier shown to be present in macrophages. In the present study, we highlight the differences in the relative subcellular localization of Rev-erbα in monocytes and macrophages. The nuclear localization of Rev-erbα in macrophages is subsequent to monocyte differentiation. Expression analysis of Rev-erbα elucidated it to be considerably more expressed in M1 phenotype in comparison with M2. Rev-erbα overexpression augments antimycobacterial properties of macrophage by keeping IL10 in a basal repressed state. Further, promoter analysis revealed that IL10 promoter harbors a Rev-erbα binding site exclusive to humans and higher order primates and not mouse, demonstrating a species barrier in its functionality. This direct gene repression is mediated by recruitment of co-repressors NCoR and HDAC3. In addition, our data elucidate that its overexpression reduced the survival of intracellular pathogen Mycobacterium tuberculosis by enhancing phagosome lysosome maturation, an event resulting from IL10 repression. Thus, these findings suggest that Rev-erbα bestows protection against mycobacterial infection by direct gene repression of IL10 and thus provide a novel target in modulating macrophage microbicidal properties.

GEI-8, a Homologue of Vertebrate Nuclear Receptor Corepressor NCoR/SMRT, Regulates Gonad Development and Neuronal Functions in Caenorhabditis elegans

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA class. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.

Nuclear receptor corepressors confer the actions of mutant thyroid hormone receptor a

Nuclear receptor corepressors confer the actions of mutant thyroid hormone receptor a

Protein Kinase A phosphorylates NCoR to enhance its nuclear translocation and repressive function in human prostate cancer cells

Protein kinase A (PKA) phosphorylates diverse protein substrates to modulate their function. In this study, we found that PKA specifically phosphorylates the RD1 (repression domain 1) domain of nuclear receptor corepressor (NCoR). We demonstrated that the Serine-70 of NCoR is identified the critical amino acid for PKA-dependent NCoR phosphorylation. Importantly, we found that PKA-dependent phosphorylation enhances the nuclear translocation of NCoR. More importantly, the activation of PKA enhanced the repressive activity of NCoR in a reporter assay and potentiated the antagonist activity in the androgen receptor (AR)-mediated transcription. Taken together, these results uncover a regulatory mechanism by which PKA positively modulates NCoR function in transcriptional regulation in prostate cancer.

Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo

Histone deacetylase 3 (HDAC3) is an epigenome-modifying enzyme that is required for normal mouse development and tissue-specific functions. In vitro, HDAC3 protein itself has minimal enzyme activity, but gains its histone deacetylation function from stable association with the conserved deacetylase activation domain (DAD) contained in nuclear receptor corepressors NCOR1 and SMRT. Here we show that HDAC3 enzyme activity is undetectable in mice bearing point mutations in the DAD of both NCOR1 and SMRT (NS-DADm), despite normal levels of HDAC3 protein. Local histone acetylation is increased, and genomic HDAC3 recruitment is reduced though not abrogated. Remarkably, the NS-DADm mice are born and live to adulthood, whereas genetic deletion of HDAC3 is embryonic lethal. These findings demonstrate that nuclear receptor corepressors are required for HDAC3 enzyme activity in vivo, and suggest that a deacetylase-independent function of HDAC3 may be required for life.

Aberrant Nuclear Repressor Coreceptor 1 Localization in Human Retinoblastoma

Background/Aims: Nuclear receptor corepressor 1 (NCoR1) is a protein complex with diverse functions in development and tumorigenesis. We investigated the pattern of expression and histopathological correlation of NCoR1 in 41 retinoblastoma tumor samples, 1 retinoblastoma cell line (WERI-Rb-1) and human retinal progenitor cells (hRPCs). Methods: Tissue sections from 41 retinoblastoma cases, the retinoblastoma cell line WERI-Rb-1 and hRPCs were stained with rabbit polyclonal anti-NCoR1 H76 antibody. Percentage and intensity of staining were classified by an ocular pathologist from 0 to 3. The paired t test was used to test for differences. Results: In the nonneoplastic retina, NCoR1 was expressed mainly in cell nuclei. The retinoblastoma tumor cells similarly had nuclear NCoR1 but also had a higher level of cytoplasmic NCoR1 expression compared to all 3 normal retinal cell layers (p < 0.002). In contrast to the normal retina, NCoR1 was mainly expressed in the cytoplasm of the proliferating WERI-Rb-1 cells. This cytoplasmic expression pattern was also seen in the undifferentiated hRPCs. Conclusions: The aberrant cytoplasmic expression of NCoR1 in retinoblastoma appears to be associated with the proliferative and/or dedifferentiated properties of retinoblastoma. Further investigation into the role of NCoR1 in retinoblastoma may provide insight into how therapeutically inhibiting its nucleocytoplasmic shuttling may affect retinoblastoma tumor biology.

SpliceArray Profiling of Breast Cancer Reveals a Novel Variant of NCOR2/SMRT That Is Associated with Tamoxifen Resistance and Control of ERα Transcriptional Activity

Gene expression profiling aimed at classifying and prognosing breast cancer has yielded signatures with little, if any, concordance. However, expression arrays used in these studies do not discriminate alternate RNA splice isoforms that vary widely in cancer and may resolve this problem. In this study, we profiled splice isoforms in a panel of tamoxifen-sensitive and -resistant cell lines, defining a novel variant (BQ323636.1) of the nuclear receptor corepressor 2 (NCOR2) that was associated with tamoxifen resistance. Overexpression of this variant in a tamoxifen-sensitive cell line induced its resistance to tamoxifen. We confirmed our initial findings from cell lines in 77 breast tumors from a Chinese cohort, where BQ323636.1 expression was higher in tamoxifen-resistant patients than tamoxifen-sensitive patients. For patients who were estrogen receptor (ER)-positive and had received tamoxifen treatment, higher BQ323636.1 expression level correlated with distant metastasis. High expression level of BQ323636.1 was found to be associated with poorer overall and disease-free survival for patients who had received tamoxifen treatment. Notably, higher BQ323636.1 versus NCOR2 wild-type ratio was also associated with negative ER and progesterone receptor (PR) status, and triple-negative status (ER-/PR-/HER2- receptor status). Mechanistic investigations showed that under conditions of tamoxifen exposure, BQ323636.1 suppressed the transcriptional activity of ERα, exhibiting promoter-regulating functions. Our findings highlight a novel splice variant of the ERα corepressor NCOR2 as a candidate biomarker in breast cancer that not only predicts tamoxifen response but may be targeted to overcome tamoxifen resistance.