Nuclear Coactivator Research Articles

Androgen receptor-positive, triple-negative breast cancer.

Androgen receptor-positive, triple-negative breast cancer.

HELZ2 Is an IFN Effector Mediating Suppression of Dengue Virus.

Flaviviral infections including dengue virus are an increasing clinical problem worldwide. Dengue infection triggers host production of the type 1 IFN, IFN alpha, one of the strongest and broadest acting antivirals known. However, dengue virus subverts host IFN signaling at early steps of IFN signal transduction. This subversion allows unbridled viral replication which subsequently triggers ongoing production of IFN which, again, is subverted. Identification of downstream IFN antiviral effectors will provide targets which could be activated to restore broad acting antiviral activity, stopping the signal to produce endogenous IFN at toxic levels. To this end, we performed a targeted functional genomic screen for IFN antiviral effector genes (IEGs), identifying 56 IEGs required for antiviral effects of IFN against fully infectious dengue virus. Dengue IEGs were enriched for genes encoding nuclear receptor interacting proteins, including HELZ2, MAP2K4, SLC27A2, HSP90AA1, and HSP90AB1. We focused on HELZ2 (Helicase With Zinc Finger 2), an IFN stimulated gene and IEG which encodes a promiscuous nuclear factor coactivator that exists in two isoforms. The two unique HELZ2 isoforms are both IFN responsive, contain ISRE elements, and gene products increase in the nucleus upon IFN stimulation. Chromatin immunoprecipitation-sequencing revealed that the HELZ2 complex interacts with triglyceride-regulator LMF1. Mass spectrometry revealed that HELZ2 knockdown cells are depleted of triglyceride subsets. We thus sought to determine whether HELZ2 interacts with a nuclear receptor known to regulate immune response and lipid metabolism, AHR, and identified HELZ2:AHR interactions via co-immunoprecipitation, found that AHR is a dengue IEG, and that an AHR ligand, FICZ, exhibits anti-dengue activity. Primary bone marrow derived macrophages from HELZ2 knockout mice, compared to wild type controls, exhibit enhanced dengue infectivity. Overall, these findings reveal that IFN antiviral response is mediated by HELZ2 transcriptional upregulation, enrichment of HELZ2 protein levels in the nucleus, and activation of a transcriptional program that appears to modulate intracellular lipid state. IEGs identified in this study may serve as both (1) potential targets for host directed antiviral design, downstream of the common flaviviral subversion point, as well as (2) possible biomarkers, whose variation, natural, or iatrogenic, could affect host response to viral infections.

Single-Molecule FRET Probes a Transient Complex that Facilitates Binding of an Intrinsically-Disordered Protein

Intrinsically-disordered proteins (IDPs) are unfolded at the native condition and fold only when attaching to their binding partners. Even though IDPs often undergo large conformational changes, which may slow the binding process, the binding kinetics are sometimes surprisingly fast, close to those of diffusion-controlled reactions. IDPs usually contain a large number of charged residues and theory predicts that the association can be facilitated by the formation of a transient complex (TC) stabilized by the electrostatic attraction between an IDP and its binding target. Although the modulation of the association rate by ionic strength of the solution has been observed for a number of IDPs, TC has not been directly probed during the course of binding. To experimentally investigate the role of TC in fast binding, we employed single-molecule Forster resonance energy transfer (FRET) spectroscopy that monitors the conformational evolution of an IDP from the unbound state to the bound complex. We studied binding of the transactivation domain (TAD) of the tumor suppressor protein p53 (negatively charged, −10) and the nuclear co-activator binding domain (NCBD) of CBP (positively charged, +6). The N- and C-termini of TAD were labeled with Alexa 647 and Alexa 488 site-specifically and molecules were immobilized on a glass surface using a biotin-neutravidin linkage to monitor binding to unlabeled NCBD in solution. Because of the fast binding kinetics, the bound state, TC, and unbound state could not be clearly resolved in binned trajectories. Instead of binning, we used the maximum likelihood method (Gopich and Szabo, JPCB(2009)) to analyze photon trajectories collected at high illumination intensity and to measure the binding kinetics (photon count rate of 50 - 100 ms−1) and the lifetime of TC (photon count rate of 500 - 1000 ms−1). We found that the association rate reaches ∼ 1 x 109 M−1s−1, which is close to the diffusion-limited rate, and the lifetime of TC is relatively long (∼100 microsecond) compared to the kinetics at low ionic strength (∼ 0.04 M). More importantly, both quantities decrease significantly with the increasing ionic strength, indicating that the destabilization of TC results in the slow association. This result demonstrates that the fast association of an IDP can be achieved by stabilizing TC through native/non-native electrostatic interactions, which would allow longer time for an IDP to interact with a target protein to fold and bind.

PRMT2 interacts with splicing factors and regulates the alternative splicing of BCL-X

Protein arginine N-methyltransferase 2 (PRMT2) functions in JAK-STAT and Wnt/β-catenin signalling pathways, serves as a nuclear receptor-dependent transcriptional co-activator, and represses NF-κB and E2F1 transcription factor activities to promote apoptosis. We have previously demonstrated that PRMT2 interacts with PRMT1 and increases its activity. Here, we reveal associations using proteomics between the PRMT2 SH3 domain and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68), a PRMT1 substrate and trans-acting factor that mediates BCL-X alternative splicing. We determined that PRMT2 interacts with SAM68 in cells and regulates its subcellular localization via the SH3 domain of PRMT2, prompting us to investigate the potential role of PRMT2 in BCL-X alternative splicing. We found that the expression of the full-length, wildtype form of PRMT2 promotes an increase in the BCL-X(L)/BCL-X(s) ratio in TNF-α or LPS stimulated cells. These results indicate that active PRMT2 may play a role during inflammation in alternative splicing regulation.

Automated Strong Cation-Exchange Cleanup To Remove Macromolecular Crowding Agents for Protein Hydrogen Exchange Mass Spectrometry.

Measuring amide hydrogen exchange (HX) of intrinsically disordered proteins (IDPs) in solutions containing high concentrations of macromolecular crowding agents would give new insights into the structure and dynamics of these proteins under crowded conditions. High concentrations of artificial crowders, required to simulate cellular crowding, introduce overwhelming interferences to mass spectrometry (MS) analysis. We have developed a fully automated, dual-stage online cleanup that uses strong cation-exchange (SCX) followed by reversed-phase desalting to remove Ficoll, a synthetic polymer, for HX-MS analysis of proteins under crowded conditions. We tested the efficiency of our method by measuring the HX-MS signal intensities of myoglobin peptides from crowded samples containing 300 g L-1 Ficoll and from uncrowded samples. Although there was loss of abundance relative to uncrowded myoglobin analyzed using conventional HX-MS, 97% coverage of the myoglobin sequence was still obtained. Control HX-MS experiments using unstructured peptides labeled at pD 4.0 under crowded and uncrowded conditions confirmed that Ficoll does not alter chemical exchange and that the same extent of HX is achieved in uncrowded solutions as in solutions containing 300 g L-1 of predeuterated Ficoll. We validated our method by measuring HX of CBP, the intrinsically disordered nuclear coactivator binding domain of CREB binding protein (UniProt CBP_MOUSE P45481 ), residues 2059-2117, at pD 6.5 under crowded and uncrowded conditions. Ficoll induced both protection and deprotection from HX in different regions of CBP, with the greatest deprotection occurring at the edges of helices. These results are consistent with previous observation of IDPs under the influence of synthetic polymers.

The microRNA network is altered in anterior cingulate cortex of patients with unipolar and bipolar depression

MicroRNAs (miRNAs) are small, non-coding RNAs acting as post-transcriptional regulators of gene expression. Though implicated in multiple CNS disorders, miRNAs have not been examined in any psychiatric disease state in anterior cingulate cortex (AnCg), a brain region centrally involved in regulating mood. We performed qPCR analyses of 29 miRNAs previously implicated in psychiatric illness (major depressive disorder (MDD), bipolar disorder (BP) and/or schizophrenia (SZ)) in AnCg of patients with MDD and BP versus controls. miR-132, miR-133a and miR-212 were initially identified as differentially expressed in BP, miR-184 in MDD and miR-34a in both MDD and BP (although none survived multiple correction testing and must be considered preliminary). In silico target prediction algorithms identified putative targets of differentially expressed miRNAs. Nuclear Co-Activator 1 (NCOA1), Nuclear Co-Repressor 2 (NCOR2) and Phosphodiesterase 4B (PDE4B) were selected based upon predicted targeting by miR-34a (with NCOR2 and PDE4B both targeted by miR-184) and published relevance to psychiatric illness. Luciferase assays identified PDE4B as a target of miR-34a and miR-184, while NCOA1 and NCOR2 were targeted by miR-34a and 184, respectively. qPCR analyses were performed to determine whether changes in miRNA levels correlated with mRNA levels of validated targets. NCOA1 showed an inverse correlation with miR-34a in BP, while NCOR2 demonstrated a positive correlation. In sum, this is the first study to demonstrate miRNA changes in AnCg in psychiatric illness and validate miR-34a as differentially expressed in CNS in MDD. These findings support a mechanistic role for miRNAs in the regulation of stress-responsive genes disrupted in psychiatric illness.

MP83-20 ENZALUTAMIDE AS AN ANDROGEN RECEPTOR INHIBITOR PREVENTS UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-20 ENZALUTAMIDE AS AN ANDROGEN RECEPTOR INHIBITOR PREVENTS UROTHELIAL TUMORIGENESIS Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, Yi Li, Satoshi Inoue, Yichun Zheng, and Hiroshi Miyamoto Takashi KawaharaTakashi Kawahara More articles by this author , Hiroki IdeHiroki Ide More articles by this author , Eiji KashiwagiEiji Kashiwagi More articles by this author , Yi LiYi Li More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Yichun ZhengYichun Zheng More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2202AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Emerging preclinical evidence suggests the critical role of androgen-mediated androgen receptor (AR) signals in the development of bladder cancer. However, little is known about the efficacy of enzalutamide, an AR signaling inhibitor that not only blocks androgen binding to AR but also prevents AR nuclear translocation, DNA binding, and coactivator recruitment, in urothelial tumorigenesis. In the current study, we aimed to assess the effects of enzalutamide as well as other anti-androgens, such as flutamide and bicalutamide, on androgen-induced neoplastic transformation of urothelial cells. METHODS An immortalized normal urothelial cell line SVHUC stably expressing wild-type AR (SVHUC-AR) was exposed to a chemical carcinogen 3-methylcholanthrene (MCA) 3 times, to induce neoplastic transformation, and subsequently cultured for 6 weeks in the presence or absence of anti-androgens. Tumorigenesis was then monitored, using plate and soft agar colony formation assays as well as mouse xenograft models. In the SVHUC-AR cells with the carcinogen challenge, quantitative real-time polymerase chain reaction analysis was also performed to determine the expression levels of tumor suppressor genes and oncogenes. RESULTS In SVHUC-AR cells, each anti-androgen was found to inhibit AR-mediated transcriptional activity. In vitro transformation showed that treatment with each anti-androgen during the process of neoplastic transformation similarly reduced the efficiency of colony formation in vitro. Compared with mock treatment, culture with enzalutamide (P = 0.028), hydroxyflutamide (P = 0.033), or bicalutamide (P = 0.038) also resulted in prevention/retardation of tumor formation in male NOD-SCID mice. Additionally, anti-androgens up-regulated the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, p27, and PTEN, and down-regulated that of several oncogenic genes, such as c-myc, cyclin D1, cyclin D3, and cyclin E, in MCA-exposed SVHUC-AR cells. However, significant effects of anti-androgens on the expression of these genes were not observed in SVHUC-AR cells without the carcinogen challenge. CONCLUSIONS Enzalutamide, flutamide, and bicalutamide similarly prevent neoplastic transformation of urothelial cells. These findings offer a potential chemopreventive approach for urothelial tumors using AR antagonists. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1088-e1089 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takashi Kawahara More articles by this author Hiroki Ide More articles by this author Eiji Kashiwagi More articles by this author Yi Li More articles by this author Satoshi Inoue More articles by this author Yichun Zheng More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract C63: Elucidating the role of AIB1 in triple-negative breast cancer cell lines

Abstract Triple negative breast cancer (TNBC) patients do not benefit from directed therapy because these tumors do not express targetable molecules; such as, hormone or growth factor receptors (ER, PR and HER2). This subtype represents 15-20% of all breast cancer cases diagnosed in women in the United States. Higher incidence of TNBC affects, particularly, young African American women compared with other women. Clinically, TNBC patients often present an advanced stage disease. These tumors exhibit an increased inflammatory signal and an aggressive phenotype that leads to an increased frequency of metastasis. These patients have a poor survival outcome. Coincidentally, expression of the amplified in breast cancer 1 (AIB1) oncogene has also been reported in higher grade, inflammatory and invasive breast cancer. AIB1 is a nuclear coactivator that regulates transcription of several genes through hormone and/or growth factor signaling. Here we demonstrate that cell lines derived from tumors in African American women representing different TNBC subtypes have varying degree of sensitive to AIB1 shRNA by lentiviral infection. AIB1 knockdown has a considerable effect on viability of all the TNBC subtypes; however, we observed the presence of a small fraction of cells that proliferate independent of AIB1. Interestingly, these cells that retain the AIB1 knockdown following sub-culture for 3-4 passages are comparable to the initial TNBC cells as regards to cell cycle and apoptosis. Intriguingly, these TNBC cells that proliferate independent of AIB1 show increase in the percentage of cells expressing CD44+/CD24- cell surface markers indicating a selection of tumor initiating cells. Concomitantly, there is a decrease in the percentage of CD44-/CD24- cells suggesting that AIB1 may regulate tumor initiating cell differentiation upon external stresses such as those caused by chemotherapeutic agents during treatment. We will further study the chemo sensitivity of TNBC cells resistant to AIB1 loss and assess whether these cells represent a model of cancer cell dormancy associated to recurrence in TNBC patients. Citation Format: Francisco R. Saenz, Anna T. Riegel. Elucidating the role of AIB1 in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C63.

Expression of the MOZ-TIF2 oncoprotein in mice represses senescence

The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16INK4a transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16INK4a transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16INK4a and p19ARF), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells.

SIRT6 deacetylates PKM2 to suppress its nuclear localization and oncogenic functions

SIRT6 (sirtuin 6) is a member of sirtuin family of deacetylases involved in diverse processes including genome stability, metabolic homeostasis, and tumorigenesis. However, the role of SIRT6 deacetylase activity in its tumor-suppressor functions is not well understood. Here we report that SIRT6 binds to and deacetylates nuclear PKM2 (pyruvate kinase M2) at the lysine 433 residue. PKM2 is a glycolytic enzyme with nonmetabolic nuclear oncogenic functions. SIRT6-mediated deacetylation results in PKM2 nuclear export. We further have identified exportin 4 as the specific transporter mediating PKM2 nuclear export. As a result of SIRT6-mediated deacetylation, PKM2 nuclear protein kinase and transcriptional coactivator functions are abolished. Thus, SIRT6 suppresses PKM2 oncogenic functions, resulting in reduced cell proliferation, migration potential, and invasiveness. Furthermore, studies in mouse tumor models demonstrate that PKM2 deacetylation is integral to SIRT6-mediated tumor suppression and inhibition of metastasis. Additionally, reduced SIRT6 levels correlate with elevated nuclear acetylated PKM2 levels in increasing grades of hepatocellular carcinoma. These findings provide key insights into the pivotal role of deacetylase activity in SIRT6 tumor-suppressor functions.

Regulation of Cardiac Transcription Factor GATA4 by Post-Translational Modification in Cardiomyocyte Hypertrophy and Heart Failure

Heart failure is a leading cause of cardiovascular mortality in industrialized countries. During development and deterioration of heart failure, cardiomyocytes undergo maladaptive hypertrophy, and changes in the cellular phenotype are accompanied by reinduction of the fetal gene program. Gene expression in cardiomyocytes is regulated by various nuclear transcription factors, co-activators, and co-repressors. The zinc finger protein GATA4 is one such transcription factor involved in the regulation of cardiomyocyte hypertrophy. In response to hypertrophic stimuli such as those involving the sympathetic nervous and renin-angiotensin systems, changes in protein interaction and/or post-translational modifications of GATA4 cause hypertrophic gene transcription in cardiomyocytes. In this article, we focus on cardiac nuclear signaling molecules, especially GATA4, that are promising as potential targets for heart failure therapy.

Folding propensity of intrinsically disordered proteins by osmotic stress.

Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.

Role of the nuclear pregnane X receptor in drug metabolism and the clinical response

The pregnane X receptor (PXR) is an orphan nuclear receptor that regulates the expression of phase I and phase II drug metabolizing enzymes and transporters involved in the absorption, distribution, metabolism, and elimination of xenobiotics. PXR is expressed predominantly in the liver and intestine and resembles cytochrome P450s (CYPs), which is a phase I drug metabolizing enzyme. It is estimated that CYP 3As and CYP2Cs metabolize > 50% of all prescription drugs. PXR upregulates gene expression of these CYPs. Therefore, PXR plays a crucial role detoxifying xenobiotics and could potentially have effects on drug-drug interactions. PXR is reportedly responsible for activating a variety of target genes through cross-talk with other nuclear receptors and coactivators at transcriptional and translation levels. Recent findings have demonstrated the regulatory role of PXR and show the potential use of a PXR antagonist during drug therapy. In addition, genetic variations in the PXR gene are associated with the pharmacological effects of several drugs, and inter-individual differences in the clinical response are likely to be understood through these PXR polymorphisms. Many approaches have been used to explain the PXR regulatory mechanisms, such as microRNA-mediated PXR post-translational regulation and diverse PXR haplotype analysis. Understanding these PXR polymorphisms may lead to improving personalized therapeutic treatments.

AMPK interacts with β-catenin in the regulation of hepatocellular carcinoma cell proliferation and survival with selenium treatment.

Selenium has received much attention as an anticancer agent, although the mechanisms of action underlying its pro-apoptotic properties remain unclear. Tumors that respond well to antioxidant treatments, such as hepatocellular carcinoma (HCC), may benefit from treatment with selenium as this compound also has antioxidant properties. Furthermore, a major oncogenic driver in HCC is the nuclear transcription co-activator, β-catenin. In the present study, we examined the mechanism by which selenium reduces survival of HCC cells, and whether this was associated with modulation of the β-catenin pathway. Hep3B cell lines and cancer cell xenografted animals were treated with selenium, and apoptotic events or signals such as AMPK, β-catenin and GSK3β were determined. Further interactions among β-catenin, glycogen synthase kinase 3β (GSK3β), and AMPK were explored by applying AMPK small interfering RNA (siRNA) or GSK3β siRNA with western blotting or immunofluorescence microscopic observation. Selenium activated AMPK, which in turn suppressed β-catenin. Selenium induced the translocation of AMPK into the nucleus and prevented the accumulation of β-catenin therein. Upon inactivation of AMPK by AMPK siRNA, selenium no longer modulated β-catenin, implying that AMPK is an upstream signal for β-catenin. We found that the binding between AMPK and β-catenin occurs in the cytosolic fraction, and therefore concluded that the cancer cell antiproliferative effects of selenium are mediated by a GSK3β-independent AMPK/β-catenin pathway, although AMPK-mediated GSK3β regulation was also observed. We primarily discovered that AMPK is a crucial regulator initiating selenium-induced inhibition of β-catenin expression. Taken together, these novel findings help to illuminate the molecular mechanisms underlying the anticancer effect of selenium and highlight the regulation of β-catenin by selenium.

Uncoupling Proteins and the Molecular Mechanisms of Thyroid Thermogenesis.

Homoeothermic species engage thermogenic components of cellular metabolism to both maintain and raise core body temperature (Tc) when exposed to cold, feeding, infection, and certain classes of drugs (sympathomimetics). Body heat production stems from two main thermogenic mechanisms: 1) obligatory thermogenesis, or the heat produced as a collective byproduct of the inefficiency of all cellular metabolic reactions, and 2) facultative thermogenesis (also referred to as adaptive or nonshivering thermogenesis [NST]), the additional heat generation required along with obligatory thermogenesis to rapidly increase body temperature. Thyroid hormone (TH) is the well-established principal endocrine regulator of both obligatory and facultative thermogenesis. Here we aim to provide a brief overview of the mechanisms by which TH regulates facultative thermogenesis and to highlight recent studies describing a role for TH in the regulation of thermogenic mitochondrial uncoupling proteins. For a broader overview of the role of the endocrine control of thermoregulation, we would refer readers to the excellent review by Silva (1). Facultative thermogenesis is controlled by both a temporary shivering (muscle contraction) component and by more sustained nonshivering mechanisms. NST is regulated primarily by both the circulating levels of TH and the acute activity of the sympathetic nervous system (SNS) and mainly occurs in brown adipose tissue (BAT) and skeletal muscle (2). The primary role of TH in NST regulation is a permissive one; it chronically sets the capacity of thermogenic and metabolic responses to SNS stimulation through its transcriptional regulation of a vast repertoire of metabolic genes (3–5). In contrast, the SNS mainly controls the acute activity of thermogenic signaling pathways culminating in the activation of heat-generating proteins downstream of -adrenergic receptors (6, 7). Owing to the fundamental importance of TH in human thermoregulation, hyperand hypothyroid patients are heat and cold intolerant, respectively (8). Similarly, hypothyroid animal models demonstrate that the TH is absolutely required for a normal thermogenic response to both cold and sympathomimetic stimulation (9). TH is synthesized in the thyroid gland and controlled by thyroid peroxidase activity that regulates the iodination, coupling, and ultimately proteolysis of tyrosine residues on thyroglobulin to release the THs, T4 and T3, into the bloodstream (10). The lesser active T4 is released from the thyroid gland at higher concentrations than the more active T3 and is locally converted to T3 in target tissues by the actions of tissue-specific deiodinases (11–13). Two genes, THRA and THRB, are responsible for the expression of distinct thyroid hormone receptors (TRs), each of which are alternatively spliced to produce multiple isoforms, TR 1, TR 2, TR 1, and TR 2, respectively (14–16). With the exception of TR 2, which does not bind T3 and functions to repress T3 actions, TR isoforms mediate distinct functions (both stimulatory and repressive) in response to and in the absence of T3 (2). Integral to their functions as transcriptional regulators, TRs bind other nuclear hormone receptors, coactivators, and corepressors, the details of which have been reviewed elsewhere (2). In addition to its transcriptional regulation, recent work has also revealed that TH may regulate cell signaling

Abstract B19: MYC induces PLD6 to suppress YAP/TAZ-dependent self-renewal of mammary stem cells

Abstract For the maintenance of a given tissue it is absolutely critical to balance proliferation and differentiation of stem cells, progenitor cells and terminally differentiated cells. Perturbations of these finely tuned processes can lead to various diseases such as cancer. One factor that has been implicated in the transition from a stem cell to a progenitor/ transit-amplifying cell is the oncogenic transcription factor MYC. Paradoxically, despite its strong pro-tumorigenic capabilities it has been shown to promote differentiation in several tissues such as the skin or the hematopoietic system. To identify critical pathways that might explain MYC's effect on the differentiation of epithelial stem cells we expressed MYC in mammary stem cells. Here, MYC induction led to a drastic reduction in sphere formation indicative of a decreased stem cell potential. Further RNA-Seq and bioinformatical analysis revealed that one of MYC's main functions in mammary stem cells is the repression of YAP/TAZ target genes. YAP and its orthologue TAZ act as nuclear co-activators and are the downstream effectors of the Hippo pathway, a recently discovered pathway with key functions in adult stem cell maintenance and growth control. Using a targeted siRNA screen for MYC target genes we were able to identify a single target gene that is absolutely essential for MYC's ability to repress YAP/TAZ, namely PLD6. PLD6 is a mitochondrial protein that promotes mitochondrial fusion, so that this MYC target gene couples a mitochondrial checkpoint to repression of YAP/TAZ. We were also able to demonstrate the relevance of this pathway in vivo using a mouse that expresses MYC in the mammary stem cell compartment. Interestingly, this pathway is also operational in human breast cancer since a high MYC signature correlates with a low YAP/TAZ signatue in breast cancer patients ultimately leading to a bad prognosis for the respective patient. In summary, we were able to identify a new pathway that couples MYC to mitochondrial biogenesis and the repression of an adult stem cell program. Citation Format: Bjoern von Eyss, Laura Annika Jaenicke, Katrin Wiese, Andreas Rosenwald, Martin Eilers. MYC induces PLD6 to suppress YAP/TAZ-dependent self-renewal of mammary stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B19.

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

MP49-16 ENZALUTAMIDE INHIBITS ANDROGEN RECEPTOR-POSITIVE BLADDER CANCER CELL GROWTH

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-16 ENZALUTAMIDE INHIBITS ANDROGEN RECEPTOR-POSITIVE BLADDER CANCER CELL GROWTH Takashi Kawahara, Hiroki Ide, Eiji Kashiwagi, Leonardo Reis, Yichun Zheng, and Hiroshi Miyamoto Takashi KawaharaTakashi Kawahara More articles by this author , Hiroki IdeHiroki Ide More articles by this author , Eiji KashiwagiEiji Kashiwagi More articles by this author , Leonardo ReisLeonardo Reis More articles by this author , Yichun ZhengYichun Zheng More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2811AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Emerging preclinical evidence suggests the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. However, little is known about the efficacy of enzalutamide, an AR signaling inhibitor that not only blocks androgen binding to AR but also prevents AR nuclear translocation, DNA binding, and coactivator recruitment, in the growth of bladder cancer cells. In this study, we compared the effects of enzalutamide and 2 other anti-androgens, flutamide and bicalutamide, on androgen-induced bladder cancer cell proliferation, migration, and invasion. METHODS MTT assay, colony formation assay, scratch wound healing assay, transwell invasion assay, real-time polymerase chain reaction, and reporter gene assay were performed in AR-positive (e.g. UMUC3, TCCSUP, 5637-AR, 647V-AR) and AR-negative (e.g. UMUC3-AR-shRNA, TCCSUP-AR-shRNA, 5637, 647V) bladder cancer lines treated with dihydrotestosterone and/or each anti-androgen. We also used a mouse xenograft model for bladder cancer. RESULTS Dihydrotestosterone increased cell viability, colony formation, cell migration, and cell invasion, indicating that endogenous or exogenous AR was functional. Enzalutamide, hydroxyflutamide, and bicalutamide showed similar inhibitory effects, without significant agonist activity, on androgen-induced cell proliferation/migration/invasion in AR-positive lines. No significant effects of dihydrotestosterone as well as anti-androgens on the growth were seen in AR-negative cells. Correspondingly, in UMUC3 cells, these anti-androgens down-regulated androgen-induced expression of MMP-2 and IL-6. Androgen-enhanced AR-mediated transcriptional activity was also blocked by each anti-androgen exhibiting marginal agonist activity. In UMUC3 xenograft-bearing mice, oral gavage treatment with each anti-androgen retarded tumor growth, and enzalutamide showed more significant suppressive effects, compared with flutamide or bicalutamide treatment. CONCLUSIONS These findings support recent findings indicating that androgens promote bladder cancer progression via the AR pathway and further suggest that anti-androgens, including enzalutamide, are of therapeutic benefit in AR-positive bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e608-e609 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takashi Kawahara More articles by this author Hiroki Ide More articles by this author Eiji Kashiwagi More articles by this author Leonardo Reis More articles by this author Yichun Zheng More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Yes-associated protein regulates the growth of human non-small cell lung cancer in response to matrix stiffness.

The Yes‑associated protein (YAP) transcriptional coactivator is recognized as a crucial regulator of human cancer. However, its involvement in human non‑small cell lung cancer (NSCLC) in response to physical cues remains unclear. In this study, substrates with different rigidity were generated in order to evaluate the role of YAP, and its upstream regulators in the Hippo pathway, in the regulation of growth of an NSCLC cell line within particular environments. It was shown that the expression of the YAP protein in SPCA-1 NSCLC cells was significantly increased when cultured on a stiff substrate compared to a soft substrate. However, the expression of phospho‑YAP protein and large tumor suppressor kinase1(LATS1) were markedly decreased after culturing on the stiff substrate. Phosphorylation of YAP by LATS1 leads to cytoplasmic retention of YAP, which inhibits its function as a nuclear transcription coactivator. The study also found that the stiff substrate promoted the growth of NSCLC cells invitro, and an increase in the transcription levels of Survivin, connective tissue growth factor, amphiregulin and Ki67, as well as a decrease in the expression level of YAP in the cytoplasm, and adecrease in p-YAP. In conclusion, the findings showed that the stiffness of the subcellular matrix altered the behavior of NSCLC cells, and that YAP regulated the growth of NSCLC cells in response to matrix stiffness, thereby suggesting a role for the Hippo‑YAP pathway in the response of NSCLC cell growth to specific microenvironments.

Cancer Cell Gene Expression Modulated from Plasma Membrane Integrin αvβ3 by Thyroid Hormone and Nanoparticulate Tetrac.

Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogs. From this receptor, thyroid hormone (l-thyroxine, T4; 3,5,3′-triiodo-l-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated non-genomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents, and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intra-cellular kinase-mediated regulation of gene expression, hormone analogs at αvβ3 have additional effects on intra-cellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation, and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.