DOCSLIB.ORG

Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Cancer Molecular and Cellular Pathobiology Research Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2, and Anna Sablina1 Abstract Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 6414–24. Ó2012 AACR. Introduction during the DNA repair process (3, 4), suggesting that phos- Genome stability is essential for the prevention of undue phatases may serve not only as negative regulators but also as cellular death and cancer development. To maintain genome active modulators of DNA damage response. integrity, cells have evolved multiple DNA repair pathways (1). Protein phosphatase 2A (PP2A) refers to a large family of One of the most powerful activators of the DNA repair response heterotrimeric Ser/Thr phosphatases. The PP2A core enzyme is double-strand breaks (DSB). consists of a catalytic C subunit and a structural A subunit. In Initiation of DSB repair is controlled by the phosphoinosi- mammals, 2 distinct genes encode closely related versions of tide 3-kinase–like kinase (PIKK) family. A wave of phosphor- both the PP2A A (Aa/PPP2R1A and Ab/PPP2R1B) and C (Ca/ ylation events radiating from PIKKs is amplified to convey the PPP2CA and Cb/PPP2CB) subunits. The AC dimer recruits a signals to a large number of substrates. Although this cascade third regulatory B subunit, which is responsible for the sub- fi has been studied in a great detail, the biologic relevance of strate speci city and function of the PP2A heterotrimeric many of these phosphorylation events and the mechanisms complex. Four unrelated families of B subunits have been identified to date: B/B55/PPP2R2A, B0/B56/PR61/PPP2R5, that control their downregulation remain unknown (2). It is 00 conceivable that Ser/Thr protein phosphatases could be B /PR72/PPP2R3, and Striatin/STRN. Approximately 100 dis- responsible for keeping proteins involved in DNA repair tinct complexes can be formed through combinatorial asso- fi response in an inactive state under normal conditions or for ciation of these subunits, and it is believed that speci c PP2A inactivating the signaling once DNA has been repaired. How- complexes mediate particular physiologic functions (5, 6). ever, phosphorylation of a number of PIKKs, including ataxia PP2A has been directly implicated in the negative regulation telangiectasia mutated (ATM), ATR, and CHK2, oscillates of DSB DNA repair proteins, including g-H2AX, ATM, CHK1, and CHK2 (2). However, consistent with the idea that protein phosphatases are not just negative regulators of DNA repair Authors' Affiliations: 1VIB Center for the Biology of Disease; Center for signaling, selective inhibition of PP2A activity impairs DNA Human Genetics, KU Leuven, Leuven, Belgium; and 2Department of repair (7–9). PP2A function is also essential for activation of Toxicology, Faculty of Preventive Medicine, Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat- cell-cycle checkpoints in response to irradiation (10, 11). One sen University, Guangzhou, China potential explanation for this apparent discrepancy is that Note: Supplementary data for this article are available at Cancer Research several distinct PP2A complexes may modulate different steps Online (http://cancerres.aacrjournals.org/). of the DNA repair process. fi Corresponding Author: Anna Sablina, CME Department, KU Leuven, O&N Here, we assessed the role of speci c PP2A complexes in I Herestraat 49, bus 602, Leuven, Belgium 3000. Phone: 32-163-30790; control of DSB repair and identify PPP2R2A as a critical Fax: 32-163-30145; E-mail: [email protected] effector of the homologous recombination (HR) repair through doi: 10.1158/0008-5472.CAN-12-1667 modulation of ATM phosphorylation. Critically, defects of HR Ó2012 American Association for Cancer Research. DNA repair in PPP2R2A-depleted cells dramatically increased 6414 Cancer Res; 72(24) December 15, 2012 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 The Role of PPP2R2A in DNA Repair their sensitivity to PARP enzyme inhibition. Finally, we show polyclonal anti-phospho-CHK1 (S296), rabbit polyclonal anti- that PPP2R2A is commonly downregulated in non–small cell phospho-CHK2 (T68), rabbit polyclonal anti-phospho BRCA1 lung carcinomas (NSCLC) and therefore propose PPP2R2A (S1524), rabbit polyclonal anti-CDC25A, rabbit polyclonal anti- status as a predictive marker for sensitivity to PARP inhibition. PPP2R2A, mouse monoclonal anti-phospho-ATM (S981; clone 10H11.E12), mouse monoclonal anti-CHK2 (clone 1C12), Materials and Methods mouse monoclonal anti-PP2A A (clone 4G7), rabbit polyclonal anti-phospho-CHK1 (S317), and rabbit monoclonal anti-phos- Plasmids and cell lines pho-CHK1 (S345; clone 133D3). Lentiviral pLA CMV N-Flag vector was used to generate Flag- tagged PPP2R2A. The pLKO.1-puro shGFP, pLKO.1-puro shLu- Neutral comet assay ciferase (shLuc), and pLKO.1-puro vectors containing short Microscope slides were precoated with a thin layer of normal hairpin RNA (shRNA) targeting specific PP2A subunits were melting point agarose. Cell suspensions were placed into a provided by the RNAi Consortium (12). The RNAi experiments tube containing low melting point agarose and transferred to a were carried out using ON-TARGETplus SMARTpools against glass microscope slide. Slides were placed in 2 mol/L NaCl, PPP2R2A and PPP2CA (Thermo Scientific). 30 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, 1% Triton X-100, Cells were cultured in Dulbecco's Modified Eagle Medium and 10% dimethyl sulfoxide (DMSO), pH 8.0, for 1 hour at 4 C. or RPMI (GIBCO) supplemented with 10% FBS and penicillin/ After lysis, the slides were rinsed for 20 minutes in solution streptomycin. Lentiviral infections were carried out as 0.3 mol/L NaOH, 1 mmol/L EDTA and were subjected to described previously (12). To induce DSBs, cells were treated electrophoresis at 0.7 V/cm and 50 mA for 20 minutes. The with bleomycin (Sigma-Aldrich) or irradiated using the linear slides were then rinsed with 1 mol/L Tris-HCl, pH 7.5 and accelerator (6 MV photons, Varian Medical Systems). dried with prechilled absolute ethanol and stained with PI (2 mg/mL). Comet images were examined at Â200 magnifica- Cell-cycle analysis tion with Nikon ECLIPSE Ti microscope. The tail moment fi Cells were washed with PBS, xed with 70% ethanol, treated values were quantified by CometScore software v1.5. with 100 mg/mL RNase A, and stained with 50 mg/mL propi- 0 dium iodide (PI). 5-Bromo-2 -deoxyuridine (BrdUrd) incorpo- Immunofluorescent microscopy ration was analyzed using the In Situ Cell Proliferation Kit Cells were plated on cover slips or mClear-96 well plates FLUOS (Roche) according to the manufacturer's protocol. Cell- (Greiner Bio-One) and fixed with 4% paraformaldehyde for 10 cycle distribution and BrdUrd immunostaining were assessed minutes at room temperature and permeabilized with ice-cold by FACSCanto (Becton-Dickinson). methanol for 5 minutes at À20C. The cells were incubated in blocking buffer 3% bovine serum albumin (BSA) in PBS for 30 Immunoblotting and immunoprecipitation minutes at room temperature and then with primary anti- Cells were resuspended in 0.5% Triton X-100, 100 mmol/L bodies. After 3 washes with 0.3% BSA in PBS, the secondary NaCl, 3 mmol/L MgCl2, 300 mmol/L sucrose, 1 mmol/L EGTA, antibody was added. The following antibodies were used: 10 mmol/L PIPES, pH 6.8, containing protease inhibitor and mouse monoclonal anti-g-H2AX (S139; Millipore, clone phosphatase inhibitor cocktails (Roche). The cell lysates JBW301) and mouse monoclonal anti-RAD51 (Abcam, clone were incubated on ice for 10 minutes and then centrifuged at 14B4). 500 Â g for 5 minutes at 4C. The supernatant and pellet were Automated Image analysis was conducted using IN Cell designated as cytoplasmic and nuclear fractions, respectively. Analyzer 2000 (GE Healthcare; Supplementary Figs. S1 and S2). The nuclear fraction was resuspended in 10 mmol/L NaCl, 5 mmol/L MgCl2, 250 mmol/L sucrose, 1 mmol/L EGTA, Quantitative real-time reverse transcription PCR 10 mmol/L Tris-HCl, pH 7.6, containing protease and phos- Total RNA was isolated using RNeasy (Qiagen), and cDNA phatase inhibitor cocktails and treated with RNase-free DNase was synthesized using the Advantage RT-PCR Kit (Clontech). I (80 mg/mL; Roche) for 30 minutes at 37C. A list of primers used for real-time quantitative PCR is pre- Immunoprecipitations were conducted 24 hours after trans- sented in Supplementary Fig. S3A. Real-time PCRs were carried fection in lysis buffer containing 50 mmol/L Tris, pH 7.4, 1% out in a Roche LightCycler-480-96 (Roche), using SYBR Green NP-40, 250 mmol/L NaCl, and proteinase inhibitor cocktail PCR Master Mix (Roche).
Load more

Recommended publications
  • Novelty Indicator for Enhanced Prioritization of Predicted Gene Ontology Annotations
    IEEE/ACM TRANSACTIONS ON COMPUTATIONAL BIOLOGY AND BIOINFORMATICS, VOL. X, NO. X, MONTHXXX 20XX 1 Novelty Indicator for Enhanced Prioritization of Predicted Gene Ontology Annotations Davide Chicco, Fernando Palluzzi, and Marco Masseroli Abstract—Biomolecular controlled annotations have become pivotal in computational biology, because they allow scientists to analyze large amounts of biological data to better understand their test results, and to infer new knowledge. Yet, biomolecular annotation databases are incomplete by definition, like our knowledge of biology, and may contain errors and inconsistent information. In this context, machine-learning algorithms able to predict and prioritize new biomolecular annotations are both effective and efficient, especially if compared with the time-consuming trials of biological validation. To limit the possibility that these techniques predict obvious and trivial high-level features, and to help prioritizing their results, we introduce here a new element that can improve the accuracy and relevance of the results of an annotation prediction and prioritization pipeline. We propose a novelty indicator able to state the level of ”newness” (or ”originality”) of the annotations predicted for a specific gene to Gene Ontology terms, and to help prioritizing the most novel and interesting annotations predicted. We performed a thorough biological functional analysis of the prioritized annotations predicted with high accuracy by using this indicator and our previously proposed prediction algorithms. The relevance
    [Show full text]
  • Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts
    Author Manuscript Published OnlineFirst on October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2 and Anna Sablina1,* 1VIB Center for the Biology of Disease; Center for Human Genetics, KULeuven, Leuven 3000 Belgium 2Department of Toxicology, Faculty of Preventive Medicine, Guangdong Provincial Key Laboratory of Food, Nutrition and Health, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China *Corresponding author information: [email protected] Contact information: Anna A. Sablina, Ph.D. CME Department, KULeuven O&N I Herestraat 49, bus 602 Leuven, Belgium 3000 Tel: +3216330790 Fax: +3216330145 Running title: The role of PPP2R2A in DNA repair Keywords: PP2A, ATM, DNA repair, cancer, PARP inhibition Conflict of interests: The authors claim no conflict of interest. - 1 - Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Reversible phosphorylation plays a critical role in DNA repair. Here we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites.
    [Show full text]
  • Patterning and Gastrulation Defects Caused by the Tw18 Lethal Are Due
    © 2017. Published by The Company of Biologists Ltd | Biology Open (2017) 6, 752-764 doi:10.1242/bio.023200 RESEARCH ARTICLE Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a Lisette Lange1,2,‡, Matthias Marks1,*,‡, Jinhua Liu1, Lars Wittler1, Hermann Bauer1, Sandra Piehl1, Gabriele Bläß1, Bernd Timmermann3 and Bernhard G. Herrmann1,4,§ ABSTRACT t haplotypes, to date only one t lethal, tw5, has been identified and The mouse t haplotype, a variant 20 cM genomic region on characterized at the molecular level (Sugimoto et al., 2012). The t w18 Chromosome 17, harbors 16 embryonic control genes identified by lethal t and related t lethals of the same complementation group 4 9 recessive lethal mutations isolated from wild mouse populations. Due (t , t ) were described about 50 years ago (Bennett and Dunn, 1960; to technical constraints so far only one of these, the tw5 lethal, has Moser and Gluecksohn-Waelsch, 1967). They cause strong been cloned and molecularly characterized. Here we report the gastrulation defects with striking overgrowth of the primitive molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal streak (PS) and bulging of cells into the amniotic cavity, die from major gastrulation defects commencing with primitive streak commencing on the seventh and prominent on the eighth to ninth formation at E6.5. We have used transcriptome and marker gene day of gestation, followed by embryonic death one day later. In analyses to describe the molecular etiology of the tw18 phenotype. We contrast, normal development requires the ingression of epiblast show that both WNT and Nodal signal transduction are impaired in the cells at the PS, epithelial-to-mesenchymal transition (EMT) and mutant epiblast, causing embryonic patterning defects and failure of migration of single mesodermal cells in between the epiblast and the primitive streak and mesoderm formation.
    [Show full text]
  • Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Ppp2r2a Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ppp2r2a conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ppp2r2a gene (NCBI Reference Sequence: NM_028032 ; Ensembl: ENSMUSG00000022052 ) is located on Mouse chromosome 14. 10 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 10 (Transcript: ENSMUST00000089230). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Ppp2r2a gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-62C15 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 4 starts from about 13.5% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 9844 bp, and the size of intron 4 for 3'-loxP site insertion: 4224 bp. The size of effective cKO region: ~666 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 10 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ppp2r2a Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats.
    [Show full text]
  • Prostaglandin A1 Inhibits the Phosphorylation of Tau Via Activating Protein Phosphotase 2A in a Michael Addition Mechanism at the Site of Cysteine 377
    Prostaglandin A1 inhibits the phosphorylation of tau via activating protein phosphotase 2A in a michael addition mechanism at the site of cysteine 377 Guo-Biao Xu Northeastern University Pei-Pei Guan Northeastern University Pu Wang ( [email protected] ) Northeastern University https://orcid.org/0000-0002-9617-5686 Research Keywords: prostaglandin A1, protein phosphotase 2A scaffold subunit A alpha, michael addition, tau, Alzheimer’s disease Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-56068/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/26 Abstract Background: Prostaglandin (PG) A1 is a metabolic product of cyclooxygenase 2 (COX-2), which potentially involved in regulating the development and progression of Alzheimer’s disease (AD). As a cyclopentenone (cy) PG, PGA1 is characterized by the presence of a chemically reactive α, β-unsaturated carbonyl. Although PGA1 is potentially involved in regulating multiple biological processes via michael addition, its specic roles in AD remained unclear. Methods: The tauP301S transgenic (Tg) mice were employed as in vivo AD models and neuroblastoma (N) 2a cells as in vitro neuronal models. By intracerebroventricular injected (i.c.v) with PGA1, the binding proteins to PGA1 are analyzed by HPLC-MS-MS. In addition, western blots are used to determine the phosphorylation of tau in PGA1 treated Tg mice in the absence or presence of okadaic acid (OA), an inhibitor of protein phosphotase (PP) 2A. Combining a synthesis of pull down assay, immunoprecipitation, western blots and HPLC-MS-MS, PP2A scaffold subunit A alpha (PPP2R1A) was identied to be activated by directly binding on PGA1 in cysteine 377-dependent manner.
    [Show full text]
  • Review of PP2A Tumor Biology and Antitumor Effects of PP2A Inhibitor LB100 in the Nervous System
    cancers Review Review of PP2A Tumor Biology and Antitumor Effects of PP2A Inhibitor LB100 in the Nervous System Jean-Paul Bryant 1 , Adam Levy 2 , John Heiss 1 and Yeshavanth Kumar Banasavadi-Siddegowda 1,* 1 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] (J.-P.B.); [email protected] (J.H.) 2 Miller School of Medicine, University of Miami, Miami, FL 33136, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-301-451-0970 Simple Summary: Central and peripheral nervous system tumors represent a heterogenous group of neoplasms which often demonstrate resistance to treatment. Given that these tumors are often refractory to conventional therapy, novel pharmaceutical regimens are needed for successfully treating this pathology. One such therapeutic is the serine/threonine phosphatase inhibitor, LB100. LB100 is a water-soluble competitive protein phosphtase inhibitor that has demonstrated antitumor effects in preclinical and clinical trials. In this review, we aim to summarize current evidence demonstrating the efficacy of LB100 as an inhibitor of nervous system tumors. Furthermore, we review the involvement of the well-studied phosphatase, protein phosphatase 2A, in oncogenic cell signaling pathways, neurophysiology, and neurodevelopment. Abstract: Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase implicated in a wide variety of regulatory cellular functions. PP2A is abundant in the mammalian nervous system, and dysregulation of its cellular functions is associated with myriad neurodegenerative Citation: Bryant, J.-P.; Levy, A.; disorders. Additionally, PP2A has oncologic implications, recently garnering attention and emerging Heiss, J.; Banasavadi-Siddegowda, as a therapeutic target because of the antitumor effects of a potent PP2A inhibitor, LB100.
    [Show full text]
  • Ppp2r2a Prostate Cancer Haploinsufficiency Is Associated
    PPP2R2A PROSTATE CANCER HAPLOINSUFFICIENCY IS ASSOCIATED WITH WORSE PROGNOSIS AND A HIGH VULNERABILITY TO B55/PP2A RECONSTITUTION THAT TRIGGERS CENTROSOME DESTABILIZATION AND INHIBITS CELL INVASION A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ziran Zhao May 2020 Examining Committee Members: Xavier Graña, PhD, Advisory Chair, Fels Institute and Dept. of Biochemistry Scott K. Shore, PhD, Fels Institute and Dept. of Biochemistry Dale S. Haines, PhD, Fels Institute and Dept. of Biochemistry M. Raza Zaidi, PhD, Fels Institute and Dept. of Biochemistry Bojana Gligorijevic, PhD, External Member, College of Engineering ABSTRACT The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through multiple phases in the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome.
    [Show full text]
  • Low Expression of PP2A Regulatory Subunit B55&Alpha
    Leukemia (2011) 25, 1711–1717 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE Low expression of PP2A regulatory subunit B55a is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients PP Ruvolo1, YH Qui1, KR Coombes2, N Zhang2, VR Ruvolo1, G Borthakur1, M Konopleva1, M Andreeff1 and SM Kornblau1 1Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA The regulation of protein kinase B (AKT) is a dynamic process by a phosphatidylinositol 3 kinase/phosphatidylinositol-depen- that depends on the balance between phosphorylation by dent kinase 1 cascade that promotes phosphorylation of the upstream kinases for activation and inactivation by depho- kinase on threonine 308 (T308) and serine 473 (S473).3,5 sphorylation by protein phosphatases. Phosphorylated AKT is A recent study has indicated that phosphorylation of AKT at commonly found in acute myeloid leukemia (AML) and confers 9 an unfavorable prognosis. Understanding the relative impor- T308 but not S473 is a poor prognostic factor for AML patients. tance of upstream kinases and AKT phosphatase in the The regulation of signal transduction is a dynamic process activation of AKT is relevant for the therapeutic targeting of dependent on the balance between phosphorylation by protein this signaling axis in AML. The B55a subunit of protein kinases and dephosphorylation by protein phosphatases.10,11 phosphatase 2A (PP2A) has been implicated in AKT depho- Aberrant activation of AKT in a leukemia cell could result either sphorylation, but its role in regulating AKT in AML is unknown.
    [Show full text]
  • Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition
    Published OnlineFirst October 18, 2012; DOI: 10.1158/0008-5472.CAN-12-1667 Cancer Molecular and Cellular Pathobiology Research Loss of PPP2R2A Inhibits Homologous Recombination DNA Repair and Predicts Tumor Sensitivity to PARP Inhibition Peter Kalev1, Michal Simicek1, Iria Vazquez1, Sebastian Munck1, Liping Chen2, Thomas Soin1, Natasha Danda1, Wen Chen2, and Anna Sablina1 Abstract Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G1 to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non–small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic. Cancer Res; 72(24); 1–11. Ó2012 AACR. Introduction during the DNA repair process (3, 4), suggesting that phos- Genome stability is essential for the prevention of undue phatases may serve not only as negative regulators but also as cellular death and cancer development.
    [Show full text]
  • PPP2R2C Loss Promotes Castration-Resistance and Is Associated with Increased Prostate Cancer-Specific Mortality
    Published OnlineFirst March 14, 2013; DOI: 10.1158/1541-7786.MCR-12-0710 Molecular Cancer Cell Death and Survival Research PPP2R2C Loss Promotes Castration-Resistance and Is Associated with Increased Prostate Cancer-Specific Mortality Eric G. Bluemn1,2, Elysia Sophie Spencer1, Brigham Mecham2,3, Ryan R. Gordon2, Ilsa Coleman2, Daniel Lewinshtein2,5, Elahe Mostaghel1,2, Xiaotun Zhang1, James Annis1,4, Carla Grandori1,2,4, Christopher Porter5, and Peter S. Nelson1,2 Abstract Metastatic prostate cancers generally rely on androgen receptor (AR) signaling for growth and survival, even following systemic androgen-deprivation therapy (ADT). However, recent evidence suggests that some advanced prostate cancers escape ADT by using signaling programs and growth factors that bypass canonical AR ligand- mediated mechanisms. We used an in vitro high-throughput RNA interference (RNAi) screen to identify pathways in androgen-dependent prostate cancer cell lines whose loss-of-function promotes androgen ligand-independent growth. We identified 40 genes where knockdown promoted proliferation of both LNCaP and VCaP prostate cancer cells in the absence of androgen. Of these, 14 were downregulated in primary and metastatic prostate cancer, including two subunits of the protein phosphatase 2 (PP2A) holoenzyme complex: PPP2R1A, a structural subunit with known tumor-suppressor properties in several tumor types; and PPP2R2C, a PP2A substrate-binding regulatory subunit that has not been previously identified as a tumor suppressor. We show that loss of PPP2R2C promotes androgen ligand depletion-resistant prostate cancer growth without altering AR expression or canonical AR-regulated gene expression. Furthermore, cell proliferation induced by PPP2R2C loss was not inhibited by the AR antagonist MDV3100, indicating that PPP2R2C loss may promote growth independently of known AR- mediated transcriptional programs.
    [Show full text]
  • A Cancer-Associated Missense Mutation in PP2A-Aα Increases Centrosome Clustering During Mitosis
    bioRxiv preprint doi: https://doi.org/10.1101/570978; this version posted March 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A cancer-associated missense mutation in PP2A-Aα increases centrosome clustering during mitosis Noelle V. Antao1, 2, Marina Marcet-Ortega2, Paolo Cifani3, Alex Kentsis3, Emily A. Foley2* 1. Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Medicine Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA. 2. Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. 3. Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA *corresponding author: [email protected] Running title: PP2A inactivation enhances centrosome clustering bioRxiv preprint doi: https://doi.org/10.1101/570978; this version posted March 7, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract A single incidence of whole-genome doubling (WGD) is common early in tumorigenesis. In addition to increasing ploidy, WGD doubles centrosome number. In the ensuing mitoses, excess centrosomes form a multipolar spindle, resulting in a lethal multipolar cell division. To survive, cells must cluster centrosomes into two poles to allow a bipolar cell division. Cancer cells are typically more proficient at centrosome clustering than untransformed cells, but the mechanism behind increased clustering ability is not well understood. Heterozygous missense mutations in PPP2R1A, which encodes the alpha isoform of the A-subunit of protein phosphatase 2A (PP2A-Aα), positively correlate with WGD.
    [Show full text]
  • Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma1
    RESEARCH ARTICLE Neoplasia . Vol. 8, No. 5, May 2006, pp. 413 – 418 413 www.neoplasia.com Fusion of the Tumor-Suppressor Gene CHEK2 and the Gene for the Regulatory Subunit B of Protein Phosphatase 2 PPP2R2A in Childhood Teratoma1 Yuesheng Jin*, Fredrik Mertens*, Carl-Magnus Kullendorff y and Ioannis Panagopoulos* *Department of Clinical Genetics, Lund University Hospital, 221 85 Lund, Sweden; yDepartment of Pediatric Surgery, Lund University Hospital, 221 85 Lund, Sweden Abstract We characterized the molecular genetic consequences aggressive subtypes of germ cell tumors, which typically have of a balanced chromosome translocation t(8;22)(p21; aneuploid karyotypes with a gain of the short arm of chro- q12), which occurred as the sole cytogenetic aberration mosome 12 as the most common feature, mature teratomas in short-term cultured cells from an intrathoracic ma- often display normal karyotype on cytogenetic analysis, and no ture teratoma in a 15-year-old girl. Fluorescence in situ consistent pattern of chromosomal gains or losses has been hybridization and reverse transcription–polymerase disclosed by comparative genomic hybridization [3,4]. Further- chain reaction disclosed that t(8;22) resulted in the more, neither in mature teratomas nor in germ cell tumors, in fusion of the genes PPP2R2A and CHEK2,withan general, has any recurrent, acquired, tumor-specific, balanced inserted fragment belonging to class I endogenous chromosome rearrangement been detected. Recently, how- retrovirus–related sequences at the junction. Sequenc- ever, a constitutional t(12;15)(q13;q25), resulting in the fusion ing of the two genes did not reveal any additional mu- of the genes SENP1 and MESDC2, was identified in a patient tation.
    [Show full text]