SIRT2 Deacetylates and Inhibits the Peroxidase Activity of Peroxiredoxin
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Published OnlineFirst August 8, 2016; DOI: 10.1158/0008-5472.CAN-16-0126 Cancer Therapeutics, Targets, and Chemical Biology Research SIRT2 Deacetylates and Inhibits the Peroxidase Activity of Peroxiredoxin-1 to Sensitize Breast Cancer Cells to Oxidant Stress-Inducing Agents Warren Fiskus1, Veena Coothankandaswamy2, Jianguang Chen3, Hongwei Ma4, Kyungsoo Ha5, Dyana T. Saenz1, Stephanie S. Krieger1, Christopher P. Mill1, Baohua Sun1, Peng Huang6, Jeffrey S. Mumm7, Ari M. Melnick8, and Kapil N. Bhalla1 Abstract SIRT2 is a protein deacetylase with tumor suppressor activ- induced by oxidative stress, as associated with increased levels ity in breast and liver tumors where it is mutated; however, the of nuclear FOXO3A and the proapoptotic BIM protein. In critical substrates mediating its antitumor activity are not fully addition, elevated levels of SIRT2 sensitized breast cancer cells defined. Here we demonstrate that SIRT2 binds, deacetylates, to arsenic trioxide, an approved therapeutic agent, along and inhibits the peroxidase activity of the antioxidant protein with other intracellular ROS-inducing agents. Conversely, anti- peroxiredoxin (Prdx-1) in breast cancer cells. Ectopic over- sense RNA-mediated attenuation of SIRT2 reversed ROS- expression of SIRT2, but not its catalytically dead mutant, induced toxicity as demonstrated in a zebrafish embryo model increased intracellular levels of reactive oxygen species (ROS) system. Collectively, our findings suggest that the tumor induced by hydrogen peroxide, which led to increased levels of suppressor activity of SIRT2 requires its ability to restrict the an overoxidized and multimeric form of Prdx-1 with activity as antioxidant activity of Prdx-1, thereby sensitizing breast a molecular chaperone. Elevated levels of SIRT2 sensitized cancer cells to ROS-induced DNA damage and cell cytotoxicity. breast cancer cells to intracellular DNA damage and cell death Cancer Res; 76(18); 5467–78. Ó2016 AACR. Introduction recognized as a tumor suppressor, because its loss in mice is associated with mammary tumors and hepatocellular carcinomas SIRT2 is a predominantly cytoplasmic member of the class III (5). Among other notable substrates deacetylated by SIRT2 are histone deacetylases (HDAC), or sirtuins, which function as þ metabolic enzymes, including glucose 6 phosphate dehydroge- NAD -dependent lysine deacetylases (1–3). Similar to HDAC6, nase (G6PD), ATP citrate lyase (ACLY), and phosphoglycerate SIRT2 colocalizes with microtubules in the cytosol and deacety- mutase (PGAM; refs. 7–9). Oxidative stress has been shown lates lysine 40 on a-Tubulin (2). During G –M phase of the cell 2 to induce SIRT2-mediated deacetylation of the transcription fac- cycle, SIRT2 is nuclear, where it deacetylates histone protein tor FOXO3a (10). This induces the transcriptional activity of residues H4K16, H3K56, and H3K18 as well as the BUB1-related FOXO3a, causing increased expression of its target genes, includ- kinase (BUBR1), thereby controlling the activity of the anaphase- ing BIM (11). In NIH3T3 cells, ectopic overexpression of SIRT2 promoting complex/cyclosome (APC/C) and normal mitotic was also shown to induce BIM and promote cell death following progression (4–6). Because of this activity, SIRT2 prevents chro- exposure to hydrogen peroxide (H O ; ref. 10). Recently, SIRT2 mosomal instability during mitosis (4, 5). SIRT2 has also been 2 2 was shown to interact with receptor-interacting protein 3 (RIP3) and deacetylate RIP1, leading to the formation of a stable RIP1/ RIP3 complex and promotion of TNFa-induced necroptosis (12). 1Department of Leukemia, The University of Texas MD Anderson Can- 2 H2O2 is a toxic byproduct of normal cellular processes in cer Center, Houston, Texas. Hyprocell LLC, Branford, Connecticut. fi 3Georgia Regents University, Augusta, Georgia. 4Department of Cell aeorobic organisms and is detoxi ed by antioxidant enzymes Biology, University of Oklahoma Health Sciences Center, Oklahoma including catalase, glutathione peroxidases, and peroxiredoxins City, Oklahoma. 5Department of Molecular Physiology, Baylor College (Prdx; refs. 13, 14). Prdxs are a family of ubiquitously expressed, 6 of Medicine, Houston, Texas. Department of Translational Molecular 22 to 27 kDa, thiol-dependent peroxidases, with a conserved Pathology, The University of Texas MD Anderson Cancer Center, Houston Texas. 7Wilmer Eye Institute and the McKusick-Nathans Insti- cysteine residue (15, 16). Prdx-1 is a two-cysteine residue member tute of Genetic Medicine, Johns Hopkins University, Baltimore, Mary- of the PRDX family of proteins (15, 16). Prdx-1 exists as a 8 land. Division of Hematology and Medical Oncology, Weill Cornell homodimer and reduces H O , utilizing thioredoxin (TRX) as Medical College, New York, New York. 2 2 the electron donor for the antioxidation (14, 16). Prdx-1 is Note: Supplementary data for this article are available at Cancer Research expressed at high levels in the cytosol of transformed cells and Online (http://cancerres.aacrjournals.org/). is further induced by oxidative stress, for example, due to exposure Corresponding Author: Kapil N. Bhalla, The University of Texas MD Anderson to H2O2, which oxidizes the conserved cysteine of Prdx-1 to Cancer Center, 1400 Holcombe Blvd, Unit 428, Houston, TX 77030. Phone: 713- sulfenic acid (15, 16). Besides its cytoprotective antioxidant 563-7308; Fax: 713-563-7308; E-mail: [email protected] function, Prdx-1 plays a role in cellular processes involving redox doi: 10.1158/0008-5472.CAN-16-0126 signaling and reactive oxygen species (ROS; refs. 17, 18). In the Ó2016 American Association for Cancer Research. current studies, we determined that SIRT2 binds and acts as a www.aacrjournals.org 5467 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 8, 2016; DOI: 10.1158/0008-5472.CAN-16-0126 Fiskus et al. deacetylase for Prdx-1; whereas knockdown (KD) of SIRT2 washed with 1Â PBS and then sonicated in the activity assay induces acetylation, ectopic overexpression of SIRT2 deacetylates, buffer. The total reaction volume of 150 mL contained 50 mmol/L and inhibits the ROS-neutralizing, antioxidant activity of Prdx-1. HEPES-NaOH buffer, Escherichia coli thioredoxin, mammalian This sensitized breast cancer cells to DNA damage and apoptosis thioredoxin reductase, and NADPH. The reaction was initiated by induced by H2O2 through a FOXO3a-BIM–mediated cell death the addition of 2 mL of 10 mmol/L H2O2. NADH oxidation was mechanism. Consistent with this, SIRT2 overexpression also monitored for 10 minutes at 340 nm. increased cell death induced by ROS-inducing agents, including arsenic trioxide (AT) and menadione. Gene transfection and interaction studies MCF7 and HEK293 cells were transiently transfected according Materials and Methods to the instructions of the manufacturer using Fugene 6 with plasmids containing scrambled oligonucleotide (control shRNA) Reagents, antibodies, and plasmids or shRNA to SIRT2 containing a 21-nucleotide sequence, corre- AT and menadione were purchased from Sigma Aldrich. All sponding to SIRT2 mRNA—50-GAAACATCCGGAACCCTTC-30, antibodies were obtained from commercial sources. Detailed as described previously (24). For interaction studies, HEK293 descriptions of the antibodies are provided in the Supplementary cells were transfected with pcDNA (control vector) with or plas- Methods. mids for HA-SIRT2 or FLAG-Prdx-1. Cell culture The breast cancer MCF7 and MDA-MB-231, as well as Comet assay HEK293 cells were obtained from ATCC. Cells were thawed, DNA damage and repair at an individual cell level was deter- passaged, and refrozen in aliquots. Cells were used within 6 mined by the comet assay as previously described (19, 20). A months of thawing or obtaining from ATCC. The ATCC utilizes detailed method is provided in the Supplementary Methods. short tandem repeat (STR) profiling for characterization and authentication of cell lines. MCF7 and HEK293 cells were ROS assay cultured in DMEM with 10% FBS and 1% penicillin/strepto- MDA-MB231 cells were grown in black 96-well plates over- mycin and passaged two to three times per week. MDA-MB-231 night at 37 C. The next day, the cells were treated with H2O2 for cells were cultured in RPMI1640 media containing 10% FBS 30 minutes at 37 C. The media were aspirated, and the cells and 1% penicillin/streptomycin and passaged two to three were washed with 1Â PBS and DCF-DA in phenol red–free times per week (19, 20). Logarithmically growing cells were media at a final concentration of 10 mmol/L was added to the exposed to the designated concentrations and exposure interval cells and incubated for 30 minutes. The dye was washed with of the drugs. Following these treatments, cells were washed free 1Â PBS and the fluorescence was read using a BioTek fluores- of the drug(s) using 1Â PBS, and pelleted prior to performing cence plate reader (19). the studies described later. Two-dimensional differential in-gel electrophoresis Immunoprecipitation of Prdx-1 and SIRT2 S100 cytosolic extracts were prepared from HEK293 vector Following treatments, cells were washed with 1Â PBS, then and SIRT2 KD cell lines. Equal amounts of protein were sub- trypsinized and pelleted. Total cell lysates were combined with jected to immunoprecipitation with anti-acetyl lysine antibody. class-specific IgG or 2 mg of anti-Prdx-1 or anti-HA (HA-SIRT2) or The proteins were eluted with glycine buffer (pH 2.7). The anti-FLAG M2 antibody and incubated with rotation overnight at proteins were then subjected to in vitro labeling with Cy-3 and 4 C. The following day, protein A beads were added and the lysate Cy-5 N-hydroxysuccinimidyl ester. Cy-2 was used as an internal bead mixture was rotated for 90 minutes 4 C. The beads were standard. The samples were subjected to isoelectric focusing washed with 1Â PBS three times and sample buffer was added. and then separated in a second dimension by SDS-PAGE. The The beads were boiled and the samples were loaded for SDS- gels were fixed, stained, and protein spots were analyzed using PAGE. Immunoblot analyses were conducted for Prdx-1, SIRT2, or GE Healthcare DeCyder software.