Nuclear Receptor Corepressor Research Articles

MRNA expression of steroidogenic enzymes, steroid hormone receptors and their coregulators in gastric cancer.

Epidemiological and experimental findings suggest that the development of gastric cancer (GC) is regulated by steroid hormones. In postmenopausal women and older men, the majority of steroid hormones are produced locally in peripheral tissue through the enzymatic conversion of steroid precursors. Therefore, using reverse transcription-quantitative polymerase chain reaction analysis, the mRNA expression of genes encoding steroidogenic enzymes, including steroid sulfatase (STS), hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1), 17β-hydroxysteroid dehydrogenase type 7 and aromatase (CYP19A1), was investigated in primary tumoral and adjacent healthy gastric mucosa from 60 patients with GC. Furthermore, the mRNA levels for estrogen receptor α, estrogen receptor β (ESR2) and androgen receptor (AR), along with their coregulators, including proline, glutamate and leucine rich protein 1, CREB binding protein, nuclear receptor coactivator 1 (NCOA1), nuclear receptor corepressor 1 (NCOR1) and nuclear receptor subfamily 2 group F member 1 (NR2F1), were investigated. Additionally, the association between the mRNA expression of these genes and the clinicopathological features of patients with GC was examined. Significantly decreased levels of STS, HSD3B1, ESR2, AR, NCOA1 and NCOR1 mRNA, in addition to significantly increased levels of CYP19A1 mRNA were demonstrated in tumoral tissue samples compared with adjacent healthy gastric tissue samples. Deregulated expression of these genes in the analyzed tissue samples was associated with certain clinicopathological features of GC, such as age and localization of the tumor. The results of the current study suggest that all of the genes analyzed are expressed in tumoral and adjacent healthy gastric mucosa. In addition, the results indicate that abnormal expression of STS, ESR2, AR, NCOA1 and NCOR1 may serve a role in the development and progression of GC, and may be associated with specific clinicopathological features in patients with GC.

Correlations between TBL1XR1 and recurrence of colorectal cancer

More than 25% localized CRC patients died from post-operative metastasis, and risk of metastasis varies among individuals due to the high heterogeneity of CRC. Therefore, figuring out potential biomarkers for disease recurrence would be invaluable to improve the follow-up efficiency and clinical treatment. Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) is a core component of the nuclear receptor corepressor complex, which functions as a repressive coregulatory factor for multiple transcription factors. The clinical significance of TBL1XR1 in CRC hasn’t been fully elucidated. In this study, we investigated the expression of TBL1XR1 in primary CRC tissues and liver metastases from TNM stage IV CRC patients, and found that its expression in primary tumor tissues was an independent prognostic factor for tumor recurrence. Thus, we enrolled another cohort including TNM stage I-III patients to further evaluate the relationship between TBL1XR1 expression and disease recurrence. Accordingly, high TBL1XR1 expression indicates poor disease-free survival of stage I-III CRC patients. Furthermore, we confirmed the importance of β-catenin signaling pathways in TBL1XR1-mediated CRC cell oncogenicity by clinical and cellular results. Our results emphasize the necessity of individual therapy decisions based on clinical biomarkers, especially for localized CRC patients who are not routinely treated with adjunctive chemotherapy.

Intestinal NCoR1, a regulator of epithelial cell maturation, controls neonatal hyperbilirubinemia

Severe neonatal hyperbilirubinemia (SNH) and the onset of bilirubin encephalopathy and kernicterus result in part from delayed expression of UDP-glucuronosyltransferase 1A1 (UGT1A1) and the inability to metabolize bilirubin. Although there is a good understanding of the early events after birth that lead to the rapid increase in serum bilirubin, the events that control delayed expression of UGT1A1 during development remain a mystery. Humanized UGT1 (hUGT1) mice develop SNH spontaneously, which is linked to repression of both liver and intestinal UGT1A1. In this study, we report that deletion of intestinal nuclear receptor corepressor 1 (NCoR1) completely diminishes hyperbilirubinemia in hUGT1 neonates because of intestinal UGT1A1 gene derepression. Transcriptomic studies and immunohistochemistry analysis demonstrate that NCoR1 plays a major role in repressing developmental maturation of the intestines. Derepression is marked by accelerated metabolic and oxidative phosphorylation, drug metabolism, fatty acid metabolism, and intestinal maturation, events that are controlled predominantly by H3K27 acetylation. The control of NCoR1 function and derepression is linked to IKKβ function, as validated in hUGT1 mice with targeted deletion of intestinal IKKβ. Physiological events during neonatal development that target activation of an IKKβ/NCoR1 loop in intestinal epithelial cells lead to derepression of genes involved in intestinal maturation and bilirubin detoxification. These findings provide a mechanism of NCoR1 in intestinal homeostasis during development and provide a key link to those events that control developmental repression of UGT1A1 and hyperbilirubinemia.

The mutational landscape of ocular marginal zone lymphoma identifies frequent alterations in TNFAIP3 followed by mutations in TBL1XR1 and CREBBP.

Ocular marginal zone lymphoma is a common type of low-grade B-cell lymphoma. To investigate the genomic changes that occur in ocular marginal zone lymphoma, we analyzed 10 cases of ocular marginal zone lymphoma using whole-genome and RNA sequencing and an additional 38 cases using targeted sequencing. Major genetic alterations affecting genes involved in nuclear factor (NF)-κB pathway activation (60%), chromatin modification and transcriptional regulation (44%), and B-cell differentiation (23%) were identified. In whole-genome sequencing, the 6q23.3 region containing TNFAIP3 was deleted in 5 samples (50%). In addition, 5 structural variation breakpoints in the first intron of IL20RA located in the 6q23.3 region was found in 3 samples (30%). In targeted sequencing, a disruptive mutation of TNFAIP3 was the most common alteration (54%), followed by mutations of TBL1XR1 (18%), cAMP response element binding proteins (CREBBP) (17%) and KMT2D (6%). All TBL1XR1 mutations were located within the WD40 domain, and TBL1XR1 mutants transfected into 293T cells increased TBL1XR1 binding with nuclear receptor corepressor (NCoR), leading to increased degradation of NCoR and the activation of NF-κB and JUN target genes. This study confirms genes involving in the activation of the NF-kB signaling pathway is the major driver in the oncogenesis of ocular MZL.

Loss of ULK1 increases RPS6KB1-NCOR1 repression of NR1H/LXR-mediated Scd1 transcription and augments lipotoxicity in hepatic cells

ABSTRACTLipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.

The molecular mechanisms and morphological manifestations of leiomyoma reduction induced by selective progesterone receptor modulators

to investigate the molecular mechanisms and morphological substrate of reduced uterine leiomyoma in patients receiving the selective progesterone receptor modulator (SPRM) ulipristal acetate for 3 months, by estimating the immunohistochemical expression of the markers steroid receptor coactivator 1 (SRC-1), nuclear receptor corepressor 1 (NCoR-1), ER, PgR, Ki-67, p16, TGF-β, and VEGF in tumor tissue. The investigation enrolled 75 women with uterine leiomyoma, menorrhagias, and anemia. Group 1 included 40 patients who were treated with ulipristal for 3 months, followed by laparoscopic myomectomy. Group 2 consisted of 35 patients who underwent surgery without previous preparation. The intra- and postoperative parameters and molecular and morphological changes in the myomatous nodules were comparatively analyzed in both groups. After 3 months of therapy initiation, menorrhagia completely ceased, myomatous nodules decreased in size (p<0.05), hemoglobin levels were elevated (p<0.01), and total intraoperative blood loss and operative time decreased in all the patients in Group 1. The morphological substrate of partial leiomyoma reduction was leiomyocyte apoptosis and dystrophy, tumor stroma sclerosis and hyalinosis with diminished Ki-67 expression and elevated p16 in the smooth muscle cells, trophic nodular tissue disorders exhibited by vascular wall sclerosis and lower VEGF and TGF-β expression, and leiomyocyte hormonal reception dysregulation that made itself evident through the reduced expression of SRC-1 with the unchanged expression of PR and ER and the maintained level of NCoR-1. The molecular mechanisms of tumor reduction involved the reduced Ki-67 expression and elevated p16, lower VEGF and TGF-β, diminished SRC-1 expression with the maintained level of PR, ER, and NCoR-1. Overall, this is suggestive of enhanced apoptosis and reduced leiomyoma proliferation and angiogenesis induced by SPRM and indicative of the expediency of using ulipristal acetate as a preoperative agent for organ-sparing surgery in reproductive-aged patients with uterine myoma, menorrhagias, and anemia.

Pathogenesis of Diet-induced Atopic Dermatitis in Hairless Mice.

Atopic dermatitis (AD) is a common pruritic chronic skin disease. AD pathogenesis remains elusive, but may involve complex interplays among skin barrier dysfunction, Th2 inflammation, and pruritus. Current treatments for AD are still limited to symptomatic therapies. We previously showed that HR-1 hairless mice fed a special diet (HR-AD) develop AD-like symptoms; however, the ingredient(s) causing dermatitis remain unclear. In this study, we examined whether the deficiency of certain polyunsaturated fatty acids (PUFAs) was involved in the diet-induced AD pathogenesis. In the serum of HR-AD-fed mice, levels of linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), as well as their metabolites, were markedly decreased. HR-AD-induced AD symptoms were significantly ameliorated by LA supplementation, and to a lesser extent by ALA supplementation. In addition, LA metabolites, such as γ-linolenic acid and arachidonic acid, had effects similar to those of LA. Further, using semi-purified custom diets, we attempted to reproduce HR-AD-induced AD symptoms. Unexpectedly, a deficiency in unsaturated fatty acids (UFAs) alone caused mild symptoms. However, several modifications of fat and carbohydrate components in the diet revealed that dietary deficiencies of UFA and cornstarch were required to fully induce severe AD symptoms. Furthermore, we examined the influence of genetic background on the development of diet-induced AD and found that a hypomorphic mutation in the hairless gene Hr, encoding a nuclear receptor (NR) corepressor, was essential for the complete development of diet-induced pruritic atopic skin. Thus, our findings suggest that certain PUFAs and NRs are new, potential therapeutic targets for treating AD.

CRISPR screen identifies the NCOR/HDAC3 complex as a major suppressor of differentiation in rhabdomyosarcoma

Dysregulated gene expression resulting from abnormal epigenetic alterations including histone acetylation and deacetylation has been demonstrated to play an important role in driving tumor growth and progression. However, the mechanisms by which specific histone deacetylases (HDACs) regulate differentiation in solid tumors remains unclear. Using pediatric rhabdomyosarcoma (RMS) as a paradigm to elucidate the mechanism blocking differentiation in solid tumors, we identified HDAC3 as a major suppressor of myogenic differentiation from a high-efficiency Clustered regularly interspaced short palindromic repeats (CRISPR)-based phenotypic screen of class I and II HDAC genes. Detailed characterization of the HDAC3-knockout phenotype in vitro and in vivo using a tamoxifen-inducible CRISPR targeting strategy demonstrated that HDAC3 deacetylase activity and the formation of a functional complex with nuclear receptor corepressors (NCORs) were critical in restricting differentiation in RMS. The NCOR/HDAC3 complex specifically functions by blocking myoblast determination protein 1 (MYOD1)-mediated activation of myogenic differentiation. Interestingly, there was also a transient up-regulation of growth-promoting genes upon initial HDAC3 targeting, revealing a unique cancer-specific response to the forced transition from a neoplastic state to terminal differentiation. Our study applied modifications of CRISPR/CRISPR-associated endonuclease 9 (Cas9) technology to interrogate the function of essential cancer genes and pathways and has provided insights into cancer cell adaptation in response to altered differentiation status. Because current pan-HDAC inhibitors have shown disappointing results in clinical trials of solid tumors, therapeutic targets specific to HDAC3 function represent a promising option for differentiation therapy in malignant tumors with dysregulated HDAC3 activity.

USP44 Is an Integral Component of N-CoR that Contributes to Gene Repression by Deubiquitinating Histone H2B

Decreased expression of the USP44 deubiquitinase has been associated with global increases in H2Bub1 levels during mouse embryonic stem cell (mESC) differentiation. However, whether USP44 directly deubiquitinates histone H2B or how its activity is targeted to chromatin is not known. We identified USP44 as an integral subunit of the nuclear receptor co-repressor (N-CoR) complex. USP44 within N-CoR deubiquitinates H2B invitro and invivo, and ablation of USP44 impairs the repressive activity of the N-CoR complex. Chromatin immunoprecipitation (ChIP) experiments confirmed that USP44 recruitment reduces H2Bub1 levels at N-CoR target loci. Furthermore, high expression of USP44 correlates with reduced levels of H2Bub1 in the breast cancer cell line MDA-MB-231. Depletion of either USP44 or TBL1XR1 impairs the invasiveness of MDA-MB-231 cells invitro and causes an increase of global H2Bub1 levels. Our findings indicate that USP44 contributes to N-CoR functions in regulating gene expression and is required for efficient invasiveness of triple-negative breast cancer cells.

The nuclear corepressor 1 and the thyroid hormone receptor β suppress breast tumor lymphangiogenesis.

Vascular Endotelial Growth Factors C and D (VEGF-C and VEGF-D) are crucial regulators of lymphangiogenesis, a main event in the metastatic spread of breast cancer tumors. Although inhibition of lymphangiogenic gene expression might be a useful therapeutic strategy to restrict the progression of cancer, the factors involved in the transcriptional repression of these genes are still unknown. We have previously shown that Nuclear Receptor Corepressor 1 (NCoR) and the thyroid hormone receptor β1 (TRβ) inhibit tumor invasion. Here we show that these molecules repress VEGF-C and VEGF-D gene transcription in breast cancer cells, reducing lymphatic vessel density and sentinel lymph node invasion in tumor xenografts. The clinical significance of these results is stressed by the finding that NCoR and TRβ transcripts correlate negatively with those of the lymphangiogenic genes and the lymphatic vessel marker LYVE-1 in human breast tumors. Our results point to the use of NCoR and TRβ as potential biomarkers for diagnosis or prognosis in breast cancer and suggest that further studies of these molecules as potential targets for anti-lymphangiogenic therapy are warranted.

Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells.

Type I IFNs are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDCs). IFN regulatory factor (IRF)7 is a critical regulator of type I IFN production when pathogens are detected by TLR 7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in pDC are still elusive. MYC is a well-known highly pleiotropic transcription factor; however, the role of MYC in pDC function is not well defined yet. To identify the role of transcription factor MYC in human pDC, we employed a knockdown technique using human pDC cell line, GEN2.2. When we knocked down MYC in the pDC cell line, production of IFN-stimulated genes was dramatically increased and was further enhanced by the TLR9 agonist CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with nuclear receptor corepressor 2/histone deacetylase 3 for its repression. In addition, activation of TLR9-mediated NF-κB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further confirming the negative role of MYC in the antiviral response by pDC. Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in cancer and autoimmune disease.

Pokemon decreases the transcriptional activity of RARα in the absence of ligand.

Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy.

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, has a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound-4 (C-4) or gene silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at promoters of TRAIL and DR5 transcriptional activator CHOP gene by dissociating KDM4A and nuclear receptor corepressor (NCoR)-HDAC complex and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.

Nuclear receptor corepressors Ncor1 and Ncor2 (Smrt) are required for retinoic acid-dependent repression of Fgf8 during somitogenesis

Retinoic acid (RA) repression of Fgf8 is required for many different aspects of organogenesis, however relatively little is known about how endogenous RA controls gene repression as opposed to gene activation. Here, we show that nuclear receptor corepressors NCOR1 and NCOR2 (SMRT) redundantly mediate the ability of RA to repress Fgf8. Ncor1;Ncor2 double mutants generated by CRISPR/Cas9 gene editing exhibited a small somite and distended heart phenotype similar to that of RA-deficient Raldh2−/− embryos, associated with increased Fgf8 expression and FGF signaling in caudal progenitors and heart progenitors. Embryo chromatin immunoprecipitation studies revealed that NCOR1/2 but not coactivators are recruited to the Fgf8 RA response element (RARE) in an RA-dependent manner, whereas coactivators but not NCOR1/2 are recruited RA-dependently to a RARE near Rarb that is activated by RA. CRISPR/Cas9-mediated genomic deletion of the Fgf8 RARE in mouse embryos often resulted in a small somite defect with Fgf8 derepression caudally, but no defect was observed in heart development or heart Fgf8 expression. This suggests the existence of another DNA element whose function overlaps with the Fgf8 RARE to mediate Fgf8 repression by RA and NCOR1/2. Our studies support a model in which NCOR1/2 mediates direct RA-dependent repression of Fgf8 in caudal progenitors in order to control somitogenesis.

Nuclear Receptor Corepressor 1 Expression and Output Declines with Prostate Cancer Progression.

Castration therapy in advanced prostate cancer eventually fails and leads to the development of castration-resistant prostate cancer (CRPC), which has no cure. Characteristic features of CRPC can be increased androgen receptor (AR) expression and altered transcriptional output. We investigated the expression of nuclear receptor corepressor 1 (NCOR1) in human prostate and prostate cancer and the role of NCOR1 in response to antiandrogens. NCOR1 protein levels were compared between matched normal prostate and prostate cancer in 409 patient samples. NCOR1 knockdown was used to investigate its effect on bicalutamide response in androgen-dependent prostate cancer cell lines and transcriptional changes associated with the loss of NCOR1. NCOR1 transcriptional signature was also examined in prostate cancer gene expression datasets. NCOR1 protein was detected in cytoplasm and nuclei of secretory epithelial cells in normal prostate. Both cytoplasmic and nuclear NCOR1 protein levels were lower in prostate cancer than in normal prostate. Prostate cancer metastases show significant decrease in NCOR1 transcriptional output. Inhibition of LNCaP cellular proliferation by bicalutamide requires NCOR1. NCOR1-regulated genes suppress cellular proliferation and mediate bicalutamide resistance. In the mouse, NCOR1 is required for bicalutamide-dependent regulation of a subset of the AR target genes. In summary, we demonstrated that NCOR1 function declines with prostate cancer progression. Reduction in NCOR1 levels causes bicalutamide resistance in LNCaP cells and compromises response to bicalutamide in mouse prostate in vivo Clin Cancer Res; 22(15); 3937-49. ©2016 AACR.

Minireview: The Link Between ERα Corepressors and Histone Deacetylases in Tamoxifen Resistance in Breast Cancer.

Approximately 70% of breast cancers express the estrogen receptor (ER)α and are treated with the ERα antagonist, tamoxifen. However, resistance to tamoxifen frequently develops in advanced breast cancer, in part due to a down-regulation of ERα corepressors. Nuclear receptor corepressors function by attenuating hormone responses and have been shown to potentiate tamoxifen action in various biological systems. Recent genomic data on breast cancers has revealed that genetic and/or genomic events target ERα corepressors in the majority of breast tumors, suggesting that the loss of nuclear receptor corepressor activity may represent an important mechanism that contributes to intrinsic and acquired tamoxifen resistance. Here, the biological functions of ERα corepressors are critically reviewed to elucidate their role in modifying endocrine sensitivity in breast cancer. We highlight a mechanism of gene repression common to corepressors previously shown to enhance the antitumorigenic effects of tamoxifen, which involves the recruitment of histone deacetylases (HDACs) to DNA. As an indicator of epigenetic disequilibrium, the loss of ERα corepressors may predispose cancer cells to the cytotoxic effects of HDAC inhibitors, a class of drug that has been shown to effectively reverse tamoxifen resistance in numerous studies. HDAC inhibition thus appears as a promising therapeutic approach that deserves to be further explored as an avenue to restore drug sensitivity in corepressor-deficient and tamoxifen-resistant breast cancers.

Abstract 4465: Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma

Abstract Embryonal rhabdomyosarcoma (ERMS), one of the most common and lethal pediatric sarcomas, is characterized by an arrest in myogenic differentiation. We have previously shown that treatment of ERMS cells with pan histone deacetylase (HDAC) inhibitors promotes myogenic differentiation. However, the underlying mechanism specific to each HDAC in ERMS remains unknown. We have completed a high-efficiency CRISPR/Cas9 gene knockout screen of class I and II HDAC genes, revealing HDAC3 as a major driver of myogenic suppression in ERMS. HDAC3-deficient ERMS cells showed robust terminal myogenic differentiation with the formation of multinucleated myotubes. Expanding on our in vitro studies, we developed a novel tamoxifen-inducible CRISPR gene-targeting system in ERMS xenografts established in immune-compromised mice. Disruption of HDAC3 in vivo significantly suppressed tumor growth by inducing myogenic differentiation of ERMS cells. By combining inducible CRISPR gene targeting with HDAC3 structure-function analysis, we showed that HDAC3 functions in concert with the nuclear receptor co-repressor (NCOR1 and NCOR2) complex to regulate gene transcription. HDAC3/NCOR binding and HDAC3 deacetylase activity are both required for suppressing myogenic differentiation in ERMS. RNA sequencing analysis of differentially expressed genes in HDAC3-deficient ERMS cells highlighted key pathways dysregulated in ERMS. Our results suggest that epigenetic silencing of the myogenic program is a major mechanism required for ERMS tumor growth. Identification of the essential interaction between HDAC3 and the NCOR transcriptional repressor complex as well as downstream genes and pathways provides important insights into the biology of ERMS tumorigenesis. Our findings highlight potential new drug targets specific to the activity of HDAC3 in order to improve treatment of ERMS patients. Citation Format: Michael Phelps, Jenna Bailey, Terra Vleeshouwer-Neumann, Eleanor Chen. Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4465.

GGNBP2 acts as a tumor suppressor by inhibiting estrogen receptor α activity in breast cancer cells.

Gametogenetin-binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2, also referred to ZFP403, has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor-binding motif. Here, we demonstrate that GGNBP2 expression is reduced in primary human breast tumors and in breast cancer cell lines, including T47D, MCF-7, LCC9, LY2, and MDA-MB-231 compared with normal, immortalized estrogen receptor α (ERα) negative MCF-10A and MCF10F breast epithelial cells. Overexpression of GGNBP2 inhibits the proliferation of T47D and MCF-7 ERα positive breast cancer cells without affecting MCF-10A and MCF10F. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)-stimulated proliferation as well as migration, invasion, anchorage-independent growth in vitro, and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity, and attenuates ER target gene expression in T47D cells. In summary, our in vitro and in vivo findings suggest that GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor corepressor to inhibit ERα activity and tumorigenesis.

Excessive gestational calorie intake in sows regulates early postnatal adipose tissue development in the offspring

Many pregnancies in the United States are associated with maternal calorie overconsumption. Few data exist that track the impact of maternal and offspring calorie consumption on the risks for obesity development. To determine the effects of maternal calorie intake during gestation on programming for adiposity in the offspring, pregnant gilts were fed either a normal (NE) or high (HE) energy diet to induce higher than normal (30 % increase) pregnancy weight gain and the profile of genes related to adipose tissue development was determined in the subcutaneous adipose tissue of the offspring. Gilts were fed the same lactation diet after farrowing and piglets were allowed to suckle from their mothers. Offspring were also fed either a normal energy (NE) or a high energy (HE) diet after weaning (3 weeks of age). Offspring were sacrificed at 48 h, 3 weeks and 3 months of age and the subcutaneous adipose tissue obtained for gene expression analysis by RT-PCR. Gilts on the HE diet had higher pregnancy weight gain and backfat thickness than those on the NE diet. Expression of adipogenic genes, such as peroxisome proliferator activated receptor (PPAR) γ and CCAAT enhancer binding protein (CEBP) α was not different between offspring from NE and HE mothers at 48 h after birth, but they were higher (P < 0.05) at 3 weeks in the offspring from HE mothers than NE. Steroid receptor coactivator 1 (SRC1) expression was higher in HE offspring at 48 h, but not different at weaning (P < 0.05). Inhibitors of wnt signaling, soluble frizzled related protein (SFRP) 4 and 5 were also higher in HE offspring at 3 weeks. The expression of PPARγ corepressors, sirtuin 1 (Sirt1, NAD-dependent deacetylase sirtuin-1) and nuclear receptor co-repressor 1 (NCoR1), was higher (P < 0.05) in HE offspring at weaning. At 3 months, there were no effects of maternal diets on offspring adipose tissue gene expression pattern, but animals on the postweaning HE diet had a higher (P < 0.05) expression of SFRP5, WNT5a, lower SFRP5/WNT5a and TNFα. Effects of maternal calorie consumption on adipose tissue genes in the early postnatal life was transient in this study. Postweaning diet was more effective in changing offspring adipose tissue gene expression pattern and adiposity in the early postnatal period.

Hepatocyte DACH1 Is Increased in Obesity via Nuclear Exclusion of HDAC4 and Promotes Hepatic Insulin Resistance.

Defective insulin signaling in hepatocytes is a key factor in type 2 diabetes. In obesity, activation ofcalcium/calmodulin-dependent protein kinase II (CaMKII) in hepatocytes suppresses ATF6, which triggers a PERK-ATF4-TRB3 pathway that disrupts insulin signaling. Elucidating how CaMKII suppresses ATF6 is therefore essential to understanding this insulin resistance pathway. We show that CaMKII phosphorylates and blocks nuclear translocation of histone deacetylase 4 (HDAC4). As a result, HDAC4-mediated SUMOylation of the corepressor DACH1 is decreased, which protects DACH1 from proteasomal degradation. DACH1, together with nuclear receptor corepressor (NCOR), represses Atf6 transcription, leading to activation of the PERK-TRB3 pathway and defective insulin signaling. DACH1 is increased in the livers of obese mice and humans, and treatment of obese mice with liver-targeted constitutively nuclear HDAC4 or DACH1 small hairpin RNA (shRNA) increases ATF6, improves hepatocyte insulin signaling, and protects against hyperglycemia and hyperinsulinemia. Thus, DACH1-mediated corepression in hepatocytes emerges as an important link between obesity and insulin resistance.