Small-molecule inhibitor targeting orphan nuclear receptor COUP-TFII for prostate cancer treatment.

The orphan nuclear receptor COUP-TFII is expressed at a low level in adult tissues, but its expression is increased and shown to promote progression of multiple diseases, including prostate cancer, heart failure, and muscular dystrophy. Suppression of COUP-TFII slows disease progression, making it an intriguing therapeutic target. Here, we identified a potent and specific COUP-TFII inhibitor through high-throughput screening. The inhibitor specifically suppressed COUP-TFII activity to regulate its target genes. Mechanistically, the inhibitor directly bound to the COUP-TFII ligand-binding domain and disrupted COUP-TFII interaction with transcription regulators, including FOXA1, thus repressing COUP-TFII activity on target gene regulation. Through blocking COUP-TFII's oncogenic activity in prostate cancer, the inhibitor efficiently exerted a potent antitumor effect in xenograft mouse models and patient-derived xenograft models. Our study identified a potent and specific COUP-TFII inhibitor that may be useful for the treatment of prostate cancer and possibly other diseases.

Elevated COUP-TFII expression in dopaminergic neurons accelerates the progression of Parkinson's disease through mitochondrial dysfunction.

Parkinson's disease (PD) is a neurodegenerative disorder featuring progressive loss of midbrain dopaminergic (DA) neurons that leads to motor symptoms. The etiology and pathogenesis of PD are not clear. We found that expression of COUP-TFII, an orphan nuclear receptor, in DA neurons is upregulated in PD patients through the analysis of public datasets. We show here that through epigenetic regulation, COUP-TFII contributes to oxidative stress, suggesting that COUP-TFII may play a role in PD pathogenesis. Elevated COUP-TFII expression specifically in DA neurons evokes DA neuronal loss in mice and accelerates the progression of phenotypes in a PD mouse model, MitoPark. Compared to control mice, those with elevated COUP-TFII expression displayed reduced cristae in mitochondria and enhanced cellular electron-dense vacuoles in the substantia nigra pars compacta. Mechanistically, we found that overexpression of COUP-TFII disturbs mitochondrial pathways, resulting in mitochondrial dysfunction. In particular, there is repressed expression of genes encoding cytosolic aldehyde dehydrogenases, which could enhance oxidative stress and interfere with mitochondrial function via 3,4-dihydroxyphenylacetaldehyde (DOPAL) buildup in DA neurons. Importantly, under-expression of COUP-TFII in DA neurons slowed the deterioration in motor functions of MitoPark mice. Taken together, our results suggest that COUP-TFII may be an important contributor to PD development and a potential therapeutic target.

Opposing Functions of BRD4 Isoforms in Breast Cancer.

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

Small molecule JQ1 promotes prostate cancer invasion via BET-independent inactivation of FOXA1.

Recent findings have shown that inhibitors targeting bromodomain and extraterminal domain (BET) proteins, such as the small molecule JQ1, are potent growth inhibitors of many cancers and hold promise for cancer therapy. However, some reports have also revealed that JQ1 can activate additional oncogenic pathways and may affect epithelial-to-mesenchymal transition (EMT). Therefore, it is important to address the potential unexpected effect of JQ1 treatment, such as cell invasion and metastasis. Here, we showed that in prostate cancer, JQ1 inhibited cancer cell growth but promoted invasion and metastasis in a BET protein-independent manner. Multiple invasion pathways including EMT, bone morphogenetic protein (BMP) signaling, chemokine signaling, and focal adhesion were activated by JQ1 to promote invasion. Notably, JQ1 induced upregulation of invasion genes through inhibition of Forkhead box protein A1 (FOXA1), an invasion suppressor in prostate cancer. JQ1 directly interacted with FOXA1 and inactivated FOXA1 binding to its interacting repressors TLE3, HDAC7, and NFIC, thereby blocking FOXA1-repressive function and activating the invasion genes. Our findings indicate that JQ1 has an unexpected effect of promoting invasion in prostate cancer. Thus, the ill effect of JQ1 or its derived therapeutic agents cannot be ignored during cancer treatment, especially in FOXA1-related cancers.

Nuclear receptors regulate alternative lengthening of telomeres through a novel noncanonical FANCD2 pathway.

Alternative lengthening of telomeres (ALT) is known to use homologous recombination (HR) to replicate telomeric DNA in a telomerase-independent manner. However, the detailed process remains largely undefined. It was reported that nuclear receptors COUP-TFII and TR4 are recruited to the enriched GGGTCA variant repeats embedded within ALT telomeres, implicating nuclear receptors in regulating ALT activity. Here, we identified a function of nuclear receptors in ALT telomere maintenance that involves a direct interaction between COUP-TFII/TR4 and FANCD2, the key protein in the Fanconi anemia (FA) DNA repair pathway. The COUP-TFII/TR4-FANCD2 complex actively induces the DNA damage response by recruiting endonuclease MUS81 and promoting the loading of the PCNA-POLD3 replication complex in ALT telomeres. Furthermore, the COUP-TFII/TR4-mediated ALT telomere pathway does not require the FA core complex or the monoubiquitylation of FANCD2, key steps in the canonical FA pathway. Thus, our findings reveal that COUP-TFII/TR4 regulates ALT telomere maintenance through a novel noncanonical FANCD2 pathway.

SRC-3 Coactivator Governs Dynamic Estrogen-Induced Chromatin Looping Interactions during Transcription.

Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.

Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer.

Alterations in both cell metabolism and transcriptional programs are hallmarks of cancer that sustain rapid proliferation and metastasis 1 . However, the mechanisms that control the interaction between metabolic reprogramming and transcriptional regulation remain unclear. Here we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We used a kinome-wide RNA interference-based screening method to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent stimulator of glycolysis 2 , is found to be a robust stimulator of SRC-3 that coregulates oestrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes the SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway and enables purine synthesis by transcriptionally upregulating the expression of the enzyme transketolase. In addition, the two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH), which are involved in purine metabolism, were identified as SRC-3 targets that may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation of SRC-3 at Ser857 increases its interaction with the transcription factor ATF4 by stabilizing the recruitment of SRC-3 and ATF4 to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumour growth in mice and prevents metastasis to the lung from an orthotopic setting, as does Ser857Ala-mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in oestrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.

Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice.

The sexual differentiation paradigm contends that the female pattern of the reproductive system is established by default because the male reproductive tracts (Wolffian ducts) in the female degenerate owing to a lack of androgen. Here, we discovered that female mouse embryos lacking Coup-tfII (chicken ovalbumin upstream promoter transcription factor II) in the Wolffian duct mesenchyme became intersex-possessing both female and male reproductive tracts. Retention of Wolffian ducts was not caused by ectopic androgen production or action. Instead, enhanced phosphorylated extracellular signal-regulated kinase signaling in Wolffian duct epithelium was responsible for the retention of male structures in an androgen-independent manner. We thus suggest that elimination of Wolffian ducts in female embryos is actively promoted by COUP-TFII, which suppresses a mesenchyme-epithelium cross-talk responsible for Wolffian duct maintenance.

Acetylation on histone H3 lysine 9 mediates a switch from transcription initiation to elongation.

The transition from transcription initiation to elongation is a key regulatory step in gene expression, which requires RNA polymerase II (pol II) to escape promoter proximal pausing on chromatin. Although elongation factors promote pause release leading to transcription elongation, the role of epigenetic modifications during this critical transition step is poorly understood. Two histone marks on histone H3, lysine 4 trimethylation (H3K4me3) and lysine 9 acetylation (H3K9ac), co-localize on active gene promoters and are associated with active transcription. H3K4me3 can promote transcription initiation, yet the functional role of H3K9ac is much less understood. We hypothesized that H3K9ac may function downstream of transcription initiation by recruiting proteins important for the next step of transcription. Here, we describe a functional role for H3K9ac in promoting pol II pause release by directly recruiting the super elongation complex (SEC) to chromatin. H3K9ac serves as a substrate for direct binding of the SEC, as does acetylation of histone H4 lysine 5 to a lesser extent. Furthermore, lysine 9 on histone H3 is necessary for maximal pol II pause release through SEC action, and loss of H3K9ac increases the pol II pausing index on a subset of genes in HeLa cells. At select gene promoters, H3K9ac loss or SEC depletion reduces gene expression and increases paused pol II occupancy. We therefore propose that an ordered histone code can promote progression through the transcription cycle, providing new mechanistic insight indicating that SEC recruitment to certain acetylated histones on a subset of genes stimulates the subsequent release of paused pol II needed for transcription elongation.

Dysregulation of nuclear receptor COUP-TFII impairs skeletal muscle development.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been shown to inhibit myogenesis and skeletal muscle metabolism in vitro. However, its precise role and in vivo function in muscle development has yet to be clearly defined. COUP-TFII protein expression level is high in undifferentiated progenitors and gradually declines during differentiation, raising an important question of whether downregulation of COUP-TFII expression is required for proper muscle cell differentiation. In this study, we generated a mouse model ectopically expressing COUP-TFII in myogenic precursors to maintain COUP-TFII activity during myogenesis and found that elevated COUP-TFII activity resulted in inefficient skeletal muscle development. Using in vitro cell culture and in vivo mouse models, we showed that COUP-TFII hinders myogenic development by repressing myoblast fusion. Mechanistically, the inefficient muscle cell fusion correlates well with the transcriptional repression of Npnt, Itgb1D and Cav3, genes important for cell-cell fusion. We further demonstrated that COUP-TFII also reduces the activation of focal adhesion kinase (FAK), an integrin downstream regulator which is essential for fusion process. Collectively, our studies highlight the importance of down-regulation of COUP-TFII signaling to allow for the induction of factors crucial for myoblast fusion.

Decreased epithelial progesterone receptor A at the window of receptivity is required for preparation of the endometrium for embryo attachment.

The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.

COUP-TFII regulates satellite cell function and muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disease caused by mutations in the dystrophin gene. Although dystrophin deficiency in myofiber triggers the disease's pathological changes, the degree of satellite cell (SC) dysfunction defines disease progression. Here, we have identified chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) hyperactivity as a contributing factor underlying muscular dystrophy in a dystrophin-deficient murine model of DMD. Ectopic expression of COUP-TFII in murine SCs led to Duchenne-like dystrophy in the muscles of control animals and exacerbated degenerative myopathies in dystrophin-deficient mice. COUP-TFII-overexpressing mice exhibited regenerative failure that was attributed to deficient SC proliferation and myoblast fusion. Mechanistically, we determined that COUP-TFII coordinated a regenerative program through combined regulation of multiple promyogenic factors. Furthermore, inhibition of COUP-TFII preserved SC function and counteracted the muscle weakness associated with Duchenne-like dystrophy in the murine model, suggesting that targeting COUP-TFII is a potential treatment for DMD. Together, our findings reveal a regulatory role of COUP-TFII in the development of muscular dystrophy and open up a potential therapeutic opportunity for managing disease progression in patients with DMD.

Dysregulation of miRNAs-COUP-TFII-FOXM1-CENPF axis contributes to the metastasis of prostate cancer.

Although early detection and treatment of prostate cancer (PCa) improves outcomes, many patients still die of metastatic PCa. Here, we report that metastatic PCa exhibits reduced levels of the microRNAsmiR-101 and miR-27a. These micro-RNAs (miRNAs) negatively regulate cell invasion and inhibit the expression of FOXM1 and CENPF, two master regulators of metastasis in PCa. Interestingly, the repression of FOXM1 and CENPF by these miRNAs occurs through COUP-TFII, a member of the orphan nuclear receptors family. Loss of miR-101 positively correlates with the increase of COUP-TFII-FOXM1-CENPF activity in clinical PCa data sets, implicating clinical relevance of such regulation. Further studies show that COUP-TFII is a critical factor controlling metastatic gene networks to promote PCa metastasis. Most importantly, this miRNA-COUP-TFII-FOXM1-CENPF regulatory axis is also involved in the development of enzalutaminde resistance. Taken together, our findings highlight the contribution of specific miRNAs through the regulation of the COUP-TFII-FOXM1-CENPF cascade in PCa metastasis and drug resistance.

Endometrial Expression of Steroidogenic Factor 1 Promotes Cystic Glandular Morphogenesis.

Epigenetic silencing of steroidogenic factor 1 (SF1) is lost in endometriosis, potentially contributing to de novo local steroidogenesis favoring inflammation and growth of ectopic endometrial tissue. In this study, we examine the impact of SF1 expression in the eutopic uterus by a novel mouse model that conditionally expresses SF1 in endometrium. In vivo SF1 expression promoted the development of enlarged endometrial glands and attenuated estrogen and progesterone responsiveness. Endometriosis induction by autotransplantation of uterine tissue to the mesenteric membrane resulted in the increase in size of ectopic lesions from SF1-expressing mice. By integrating the SF1-dependent transcriptome with the whole genome binding profile of SF1, we identified uterine-specific SF1-regulated genes involved in Wingless and Progesterone receptor-Hedgehog-Chicken ovalbumin upstream promoter transcription factor II signaling for gland development and epithelium-stroma interaction, respectively. The present results indicate that SF1 directly contributes to the abnormal uterine gland morphogenesis, an inhibition of steroid hormone signaling and activation of an immune response, in addition to previously postulated estrogen production.

MPC1, a key gene in cancer metabolism, is regulated by COUPTFII in human prostate cancer.

Mitochondrial pyruvate carrier 1 (MPC1) and MPC 2 form a transporter complex in cells to control pyruvate transportation into mitochondria. Reduced expression of MPC1 disrupts the transporter function, induces metabolic shift to increase glycolysis, and thus plays important roles in several diseases, including cancer. However, the role of MPC1 in prostate cancer and the underlying mechanism causing the down-regulation of MPC1 in tumor cells remain to be defined. Here, we show that MPC1 serves as a critical regulator of glycolysis in prostate cancer cells, which in turn controls cancer cell growth, invasion, and the tumorigenic capability. More importantly, we identified that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a steroid receptor superfamily member, transcriptionally regulates the expression of MPC1. We further demonstrate that COUP-TFII, which is upregulated in the prostate cancer patient, regulates MPC1 and glycolysis to promote tumor growth and metastasis. Our findings reveal that COUP-TFII represses MPC1 expression in prostate cancer cells to facilitate a metabolism switch to increase glycolysis and promote cancer progression. This observation raises an intriguing possibility of targeting COUP-TFII to modulate cancer cell metabolism for prostate cancer intervention.

Choose your destiny: Make a cell fate decision with COUP-TFII.

Cell fate specification is a critical process to generate cells with a wide range of characteristics from stem and progenitor cells. Emerging evidence demonstrates that the orphan nuclear receptor COUP-TFII serves as a key regulator in determining the cell identity during embryonic development. The present review summarizes our current knowledge on molecular mechanisms by which COUP-TFII employs to define the cell fates, with special emphasis on cardiovascular and renal systems. These novel insights pave the road for future studies of regenerative medicine.

Estrogen Receptor ß Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis.

Alterations in estrogen-mediated cellular signaling play an essential role in the pathogenesis of endometriosis. In addition to higher estrogen receptor (ER) ß levels, enhanced ERß activity was detected in endometriotic tissues, and the inhibition of enhanced ERß activity by an ERß-selective antagonist suppressed mouse ectopic lesion growth. Notably, gain of ERß function stimulated the progression of endometriosis. As a mechanism to evade endogenous immune surveillance for cell survival, ERß interacts with cellular apoptotic machinery in the cytoplasm to inhibit TNF-α-induced apoptosis. ERß also interacts with components of the cytoplasmic inflammasome to increase interleukin-1ß and thus enhance its cellular adhesion and proliferation properties. Furthermore, this gain of ERß function enhances epithelial-mesenchymal transition signaling, thereby increasing the invasion activity of endometriotic tissues for establishment of ectopic lesions. Collectively, we reveal how endometrial tissue generated by retrograde menstruation can escape immune surveillance and develop into sustained ectopic lesions via gain of ERß function.

Increased COUP-TFII expression in adult hearts induces mitochondrial dysfunction resulting in heart failure.

Mitochondrial dysfunction and metabolic remodelling are pivotal in the development of cardiomyopathy. Here, we show that myocardial COUP-TFII overexpression causes heart failure in mice, suggesting a causal effect of elevated COUP-TFII levels on development of dilated cardiomyopathy. COUP-TFII represses genes critical for mitochondrial electron transport chain enzyme activity, oxidative stress detoxification and mitochondrial dynamics, resulting in increased levels of reactive oxygen species and lower rates of oxygen consumption in mitochondria. COUP-TFII also suppresses the metabolic regulator PGC-1 network and decreases the expression of key glucose and lipid utilization genes, leading to a reduction in both glucose and oleate oxidation in the hearts. These data suggest that COUP-TFII affects mitochondrial function, impairs metabolic remodelling and has a key role in dilated cardiomyopathy. Last, COUP-TFII haploinsufficiency attenuates the progression of cardiac dilation and improves survival in a calcineurin transgenic mouse model, indicating that COUP-TFII may serve as a therapeutic target for the treatment of dilated cardiomyopathy.

Estrogen receptor-α in medial amygdala neurons regulates body weight.

Estrogen receptor-α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded-1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight-lowering effects of a glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug-mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.