SIRT2 Deacetylates and Inhibits the Peroxidase Activity of Peroxiredoxin

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. 2016 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 2016 American Association for Cancer Research. current studies, we determined that SIRT2 binds and acts as a

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deacetylase for Prdx-1; whereas knockdown (KD) of SIRT2 washed with 1 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 1 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 1 PBS and the fluorescence was read using a BioTek fluores- of the drug(s) using 1 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 1 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 1 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. The protein spots of interest acetyl lysine, as described previously (19, 20). were subjected to automated in-gel tryptic digestion and MALDI/MS/MS spectra were performed with 4800 Proteomics SDS-PAGE and immunoblot analyses Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosys- fi Seventy- ve micrograms of total cell lysates were used for SDS- tems; refs. 24, 25). PAGE and immunoblot analyses, as described previously (21). High molecular weight oligomer formation Confocal microscopy MDA-MB-231, MCF7 and HEK293 vector, and SIRT2 O/E cells MCF7 or MDA-MB231 cells were labeled with immunofluo- were exposed to the indicated concentration of H O for differ- rescence-tagged antibodies, as described previously (22). 2 2 ent time points and the lysates were resolved in a 8%native PAGE Assessment of propidium iodide–positive cells electrophoresis. The formation of high molecular weight oligo- Untreated or drug-treated cells were stained with propidium mers were visualized with antioxidized-Prdx-1 or anti-Prdx-1 In vivo iodide (PI), and the percentage of PI-positive cells was determined antibody, as described previously (18, 26). overoxidation by flow cytometry, as described previously (21, 22). of Prdx-1 was determined, as described previously (27).

Peroxiredoxin activity assay In vitro chaperone activity of Prdx-1 The Prdx activity assay was carried out according to manufac- HEK293, MDA-MB-231, and MCF7 cells stably transfected with turer's instructions (Redoxica) and as previously described (23). vector, or SIRT2 cDNA were treated with H2O2 for 30 minutes, Brieﬂy, cells were collected by trypsinization. The cells were respectively, and the cells were harvested and lysed with native

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SIRT2 Regulates Sensitivity to ROS in Breast Cancer

lysis buffer. Protein concentrations were measured using a BCA the FLAG-tagged Prdx-1 and HA-tagged SIRT2 into HEK293 cells. Kit. Endogenous oligomers and multimers of Prdx-1 protein were The cells were lysed, and the anti-HA antibody immunoprecipi- immunoprecipitated using Dynabeads M-280 sheep anti-mouse tates were then immunoblotted with the anti-FLAG and anti-HA IgG (Invitrogen) and anti-Prdx-1 (LF-MA0214; AbFrontier) antibodies. As shown in Fig. 2A, the epitope-tagged Prdx-1 coim- according to the manufacturer's instructions. The immunopreci- munoprecipitated with the epitope-tagged SIRT2. We further pitated Prdx-1 and Dynabeads were used for chaperone activity confirmed that the endogenous Prdx-1 also coimmunoprecipi- assay as described previously (18, 26). Briefly, each reaction tated with the FLAG-tagged SIRT2 introduced into MDA-MB-231 contained 2 mmol/L malate dehydrogenase (MDH) and immu- (Fig. 2B) and MCF7 cells (Fig. 2C). Next, we determined whether noprecipitated chaperone with Dynabeads in 200 mLof50 the genetic KD or chemical inhibition of SIRT2 affects the acet- mmol/L HEPES-KOH buffer (pH 7.5). The chaperone activity ylation of the endogenous Prdx-1 in MCF7 cells. Figure 2D was monitored by measuring the absorbance (320 nm) in a demonstrates that chemical inhibition of the catalytic activity of BioTeck SynergyMx plate reader at 43C for 2 hours. SIRT2 by nicotinamide (NA) or shRNA-mediated 90% KD of SIRT2-induced lysine acetylation of Prdx-1. We next performed the reverse immunoprecipitation with anti-acetylated lysine in Zebrafish studies cells with ectopic overexpression or KD of SIRT2. Figure 2E shows Wild-type (AB) zebrafish were maintained using standard that ectopic overexpression of SIRT2 (shown above in Fig. 2B) procedures (28). Zebrafish embryos and larvae were obtained by deacetylates Prdx-1, resulting in attenuated levels of the immu- natural mating. Morpholino oligonucleotides were injected into noprecipitated, acetylated Prdx-1. Conversely, SIRT2 KD caused yolk at the one-cell stage using an IM300 microinjector (Nar- an increase in the levels of the immunoprecipitated acetylated ishige; ref. 29). The SIRT2 MO (Genetools LLC) were designed Prdx-1 (Fig. 2E). against the splice-donor sites of exon 6 of SIRT2: 50-TATG- TAAAGTCAGACCTGTTTGTG-30. SIRT2 MO was injected (0.5 nL) Levels of SIRT2 affect Prdx-1 acetylation and its into the yolk of 1-cell-stage zebrafish embryos at a final quantity of antioxidant activity 4 ng. To validate the KD, total RNA was extracted from 48 hpf Next, we determined whether ectopic overexpression or acti- embryos using TRIzol reagent. Two hundred nanograms of total vation of SIRT2 or KD of SIRT2 by shRNA perturbs acetylation RNA was reverse transcribed using a High-Capacity Reverse Tran- of the endogenous Prdx-1. For this, we ectopically overexpressed scription Kit (Applied Biosystems) following manufacturer's pro- or knocked-down SIRT2 by shRNA in MCF7 and MB-231 cells tocol. qRT-PCR was carried out using SYBR green. For evaluation (Fig. 3A). We also ectopically expressed the catalytically inactive of toxicity, 48 hpf embryos injected with SIRT2 MO or a 5-bp mutant form of SIRT2 (H187A), created by site-directed muta- mismatch control were treated with the indicated concentrations genesis (10), in MB-231 cells (Fig. 3A and B). First, we confirmed of H O . At the end of 48 hours of treatment no mortality was 2 2 that the ectopic expression of SIRT2, but not of the mutant SIRT2, observed but there were morphologic abnormalities, as described increased the intracellular lysine deacetylase activity in MB-231 previously (29). The embryos were placed in tricaine solution and and MCF7 cells (Supplementary Fig. S1 and data not shown). imaged using an epifluorescence microscope. For estimation of However, enforced alterations in the levels of SIRT2 neither the ROS, 48 hpf embryos injected with the SIRT2 MO or 5-bp affected the levels of Prdx-1, nor led to any change in the levels mismatch control were treated with H O for 30 minutes. The 2 2 of several other HDACs, for example, SIRT3, SIRT6, and HDAC6, embryos were then incubated with 50 mmol/L DCF-DA (Invitro- or of the antioxidant proteins, including superoxide dismutase gen). Individual embryos were transferred to each well of a 96- and catalase (Supplementary Fig. S2A and data not shown). well plate and read using a plate reader. At least six embryos per Ectopically expressed wild-type and mutant SIRT2 were localized experimental condition were used. to the cytoplasm of MB-231 and MCF7 cells (Fig. 3B and Sup- plementary Fig. S2B). As shown in Fig. 3C, compared with the Results MB-231 transduced with the vector alone, by deacetylating Prdx- SIRT2 binds and deacetylates Prdx-1 1, ectopically overexpressed SIRT2 significantly inhibited the Among the known cytosolic targets of deacetylation by SIRT2 is antioxidant activity of Prdx-1 (P ¼ 0.01). Conversely, MB-231 a-tubulin (2). As shown in Fig. 1A, KD of SIRT2 with two separate cells exhibiting shRNA-mediated KD of SIRT2 showed a signif- shRNAs stably transduced into HEK-293 cells induced the acet- icant increase in the antioxidant activity of the resulting hyper- ylation of a-tubulin, without altering the levels of the total acetylated Prdx-1 (Fig. 3C). Here, the antioxidant activity of the a-tubulin. In addition, lysates from cells transduced with the deacetylated or hyperacetylated Prdx-1 in the cellular protein control shRNA or SIRT2 shRNA were also immunoblotted with extract was assayed by estimating its reducing effect on H2O2- anti-acetyl-lysine antibody, again demonstrating increased acet- mediated NADPH oxidation, which was compared with the ylation of several proteins, including a-tubulin and histone H3 antioxidant activity of the recombinant Prdx-1 in the same assay (Fig. 1B). We next performed the two-dimensional differential in- (Fig. 3C). As compared with the control MB-231 (transduced with gel electrophoresis (DIGE) analysis on the S100 cytosolic extracts vector alone), treatment of MB-231 cells with ectopic overexpres- from HEK293 shRNA control and SIRT2 KD cell lines. Figure 1C sion of SIRT2 with 500 mmol/L of H2O2 resulted in significantly demonstrates a representative two-dimensional-gel image show- increased intracellular levels of ROS (P ¼ 0.01; Fig. 3D). Exposure ing the protein spots exhibiting more than a 2-fold difference in to H2O2 caused overoxidation of the active sulfhydryl residues in the mobility between the vector control and SIRT2 KD–treated Prdx-1 to sulfinic or sulfonic acid, which was detected by utilizing samples. One of the spots demonstrating a 2.15-fold change in its the antibody that recognizes the overoxidized cysteine in Prdx-1 mobility was identified by mass spectrometry to be Prdx-1. We (27), in MB-231 cells overexpressing wild-type SIRT2 but not also identified phosphoglycerate kinase 1 (PGK1) and MDH (Fig. those expressing the catalytically dead mutant form of SIRT2 1C). To confirm that SIRT2 interacts with Prdx-1, we introduced (Fig. 3E). Following exposure of the MB-231 cells expressing the

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HEK293 A B HEK293 SIRT2 shRNA clone SIRT2 shRNA 1 2 NT shRNA Control shRNA SIRT2 1.0 0.18 0.18 Ac. α-Tubulin

1.0 1.5 1.6 Anti- α-Tubulin Ac. lysine α-Tubulin Hyperacetylated proteins β-Actin

Histone H3

150 100 75

50 MDH, [+ 2.12-fold]

25 PGK1, [+ 2.15-fold] Peroxiredoxin 1,[+ 2.15-fold]

Figure 1. KD of SIRT2 by shRNA induces acetylation of proteins including Prdx-1. A, HEK293 cells were stably transfected with control shRNA or SIRT2 shRNA constructs. Total cell lysates were prepared from the cell lines and immunoblot analyses were performed for SIRT2, acetylated a-Tubulin, and a-Tubulin. The expression levels of b-actin served as the loading control. B, immunoblot analysis of total acetylated lysine in cell lysates from control shRNA or SIRT2 shRNA–transfected HEK293 cells. C, S100 cytosolic extracts were prepared from HEK293 vector and SIRT2-KD cell lines and 2D DIGE was performed. A representative 2D-gel image is presented. Protein spots exhibiting more than a 2-fold difference in mobility in SIRT2-KD samples compared with vector control were identiﬁed by mass spectrometry. Arrows, the location of Prdx-1, MDH, and phosphoglycerate kinase 1 (PGK1).

catalytically dead mutant form of SIRT2 to H2O2, no increase in stress switches Prdx-1 back from a chaperone to a low molecular the levels of the oxidized Prdx-1 was observed (Fig. 3E). There- weight peroxidase function (18, 30). Next, we determined wheth- fore, the reduced antioxidant activity of the de-acetylated Prdx-1 er in SIRT2-overexpressing cells expressing deacetylated Prdx-1, was associated with increased levels of ROS as well as increase in the latter is overoxidized due to increased intracellular levels of the oxidized form of Prdx-1, whereas in the catalytically dead ROS, and whether Prdx-1 would form multimers and exhibit mutant, Prdx-1 was relatively resistant to oxidation. Collectively increased chaperone activity toward one of its known substrate these ﬁndings highlight that, although the antioxidant activity of proteins, MDH (31). Figure 4A and Supplementary Fig. S3 dem- Prdx-1 regulates intracellular levels of H2O2, high levels of onstrate that following exposure to 500 mmol/L of H2O2 for H2O2 in turn oxidize and regulate the antioxidant activity of 30 minutes to 4 hours, as compared with their respective controls, Prdx-1 (15, 16). HEK293 and MB-231 cells overexpressing SIRT2 exhibited The cytosolic 2-Cys Prdx-1 has dual function as a peroxidase increased levels of the high molecular weight multimers of and molecular chaperone (18). Upon exposure to oxidative stress, Prdx-1 and their oxidized counterparts (right and left panel), as Prdx-1 transitions to high molecular weight oligomers and func- detected by anti-Prdx-1 and anti-oxidized Prdx-1 antibody, tions as a molecular chaperone (30). Removal of the oxidative respectively. Prdx-1 displays a robust capacity to suppress the

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A HEK293 B MDA-MB-231 C

+ – + HA-SIRT2 MCF7/SIRT2 IgG – + + FLAG-Prdx-1 IP:FLAG IP: HA-SIRT2 IB: IB: Input IgG FLAG Prdx-1 IgG L.C. Prdx-1 HA SIRT2 IP: FLAG SIRT2 Total cell lysate input Vector FLAG- SIRT2 input IgG Vector FLAG-SIRT2 IgG Vector FLAG-SIRT2 IB: IgG L.C. FLAG Prdx-1 Prdx-1 HA SIRT2 SIRT2

D E MDA-MB-231 MCF7

IP: Prdx-1 IP:Ac K IgG Control NA Control + KD SIRT2 Input IB: IB: Ac. lysine input Vector SIRT2 O/E input SIRT2 KD Input IgG Vector SIRT2 O/E IgG SIRT2 KD IgG Vector SIRT2 O/E SIRT2 KD 1.0 2.4 2.0 IgG Light chain Prdx-1 Prdx-1

Total cell lysates 1.0 0.74 2.03 SIRT2 1.0 0.9 0.1 Prdx-1 1.0 1.1 1.1 β-Actin

Figure 2. SIRT2 interacts with Prdx-1. A, cells lysates from HEK293 cells expressing FLAG-tagged Prdx-1 and HA-tagged SIRT2 were immunoprecipitated with anti-HA antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-FLAG and anti-HA antibodies. Total cell lysates were subjected to immunoblot analyses with anti-FLAG and anti-HA antibodies. B, MDA-MB-231 vector and FLAG-SIRT2–overexpressing cells were lysed and immunoprecipitated with anti-FLAG antibody. Immunoblot analyses were performed for Prdx-1 and SIRT2 on the immunoprecipitates. Position of the IgG light chain (LC) is indicated with an arrow. C, MCF7 FLAG-SIRT2–overexpressing cells were lysed and immunoprecipitated with anti-FLAG M2 antibody–conjugated beads. Immunoblot analyses were performed for Prdx-1 and SIRT2 on the immunoprecipitates. Vertical line, a repositioned gel lane. D, cell lysates from MCF7 cells treated with 1 mmol/L of NA or transfected with SIRT2 shRNA were subjected to immunoprecipitation with anti-Prdx-1 antibody. Immunoblot analysis was performed with anti-acetyl lysine and anti-Prdx-1 antibodies on the immunoprecipitates. Total cell lysates were also immunoblotted with anti-SIRT2, anti-Prdx-1, and b-actin antibodies. Values underneath the blots indicate densitometry analysis. E, MDA-MB-231 cells transfected with vector, ectopic overexpression of SIRT2, or KD of SIRT2 were lysed and immunoprecipitated with anti-acetyl-lysine antibody. Immunoblot analysis was performed for Prdx-1 on the immunoprecipitates. Values underneath the blots indicate densitometry analysis.

misfolding of MDH. Thus, the increased chaperone function of in the SIRT2-overexpressing versus the vector control MB-231 the oxidized Prdx-1 multimers in SIRT2 overexpressing versus the cells. This was estimated in the nuclei by confocal immunoﬂu- vector control HEK293 and MB-231 cells exposed to H2O2 orescence microscopy, as well as in the cell lysates by immunoblot resulted in reduced absorbance at 320 nm (less light scattering analysis (Fig. 5A and B). We also determined the DNA damage of misfolded MDH), which was due to improved folding of MDH and repair at an individual cell level by the Comet assay (19, 20). (Fig. 4B). Similar effects were also observed in SIRT2-overexpres- Treatment with H2O2 induced more DNA damage, determined by sing MCF7 versus the vector control cells exposed to lower con- estimating the length of the comet tails, in the SIRT2 overexpres- centrations of H2O2 for 30 minutes (Fig. 4C and D). sing versus the vector control MB-231 cells (Fig. 5C). Although a higher percentage of untreated cells exhibited shorter comet tails Increased ROS and DNA damage is due to inactivation of Prdx-1 and lower tail movement, treatment with H2O2 dose-dependently from SIRT2-mediated deacetylation increased the percentage of cells with longer comet tails and We next determined whether reduced antioxidant activity of higher tail movement, more so in the SIRT2 overexpressing versus Prdx-1 and higher intracellular ROS levels in the SIRT2-over- the vector control MB-231 cells (Fig. 5C and D). In contrast, expressing cells would result in increased DNA damage, especially exposure to H2O2 induced less DNA damage represented by following exposure to H2O2. Figure 5A and B demonstrate that higher % of cells with shorter tail movement in MB-231 cells treatment with H2O2 induced higher intracellular levels of expressing either the catalytically dead mutant SIRT2 or KD of g-H2AX, signifying an increase in the DNA damage and response, SIRT2 (Fig. 5C and D).

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MDA-MB-231 A MDA-MB-231 MCF7 B SIRT2 Vector SIRT2 O/E Mutant

FLAG- SIRT2 Vector SIRT2 KD SIRT2 Vector SIRT2 O/E SIRT2 KD SIRT2 SIRT2 O/E Control shRNA SIRT2 Mutant Control shRNA SIRT2 DAPI

β-Actin

MERGE C 3,000 MDA-MB-231 2,500 P < 0.05 D 600 2,000 MDA-MB-231 Vector 500 MDA-MB-231 SIRT2 O/E 1,500 P = 0.01 400 P = 0.01 1,000

μmoles of NADH 300 500

oxidized/min/mg protein 200

0 Control) of (% ROS Levels RFU ROS Levels 100 0 0100500

H2O2 (mmol/L), 2 hours E MDA-MB-231

0 4 Hours 8 Hours 500 μmol/L, H2O2 Vector Vector Vector SIRT2 O/E SIRT2 O/E SIRT2 O/E SIRT2-mutant SIRT2-mutant SIRT2-mutant Ox-Prdx-1 Prdx-1 α-Tubulin

Figure 3. SIRT2 deacetylates Prdx-1 and decreases its activity. A, MDA-MB-231 and MCF7 cells were stably transfected with vector or SIRT2 O/E and SIRT2 (H187A) mutant constructs or with control shRNA and SIRT2 shRNA constructs as indicated. Total cell lysates were prepared from the cell lines and immunoblot analyses were performed for the expression levels of SIRT2 and of b-actin. B, MDA-MB-231–overexpressing vector, SIRT2, or SIRT2 (H187A) mutant proteins were ﬁxed, permeabilized, and blocked with 3% BSA. The expression of FLAG was detected by immunoﬂuorescent staining with FLAG M2 antibody followed by staining with Alexa Fluor 555–conjugated anti-mouse secondary antibody. Nuclei were stained with DAPI. Images were acquired using an LSM-510 Meta confocal microscope

(Carl Zeiss) with a 63/1.2 NA oil immersion lens. C, total lysates from MDA-MB-231 vector, SIRT2 O/E, or SIRT2 KD cells were utilized to determine H2O2 reducing activity and the percentage change in Prdx-1 activity (NADPH oxidized/min/mg protein). Recombinant Prdx-1 (3 mg) was used as a positive control for the assay. D, MDA-MB-231 vector or SIRT2 O/E cells were grown in 96-well plates. The next day, the cells were treated with the indicated concentrations of H2O2 for 2 hours at 37C. The media were aspirated and cells were washed with 1 PBS. DCF-DA (in phenol red–free media) was added to the cells at a ﬁnal concentration of 10 mmol/L and incubated for 30 minutes at 37C. The excess dye was removed by washing with 1 PBS and the ﬂuorescence was read at 485 nm using a BioTek plate reader.

E, MDA-MB-231 vector, SIRT2 O/E, and SIRT2 (H187A)-mutant expressing cells were treated with H2O2 for 4 and 8 hours, as indicated. Following this, total cell lysates were prepared and immunoblot analyses were performed for oxidized-Prdx-1 and total Prdx-1. The expression levels of a-Tubulin served as loading control.

FOXO3A and BIM involvement in increased cell death due to ment with H2O2 further increased the levels of FOXO3A in oxidative stress in SIRT2-overexpressing breast cancer cells the nucleus of SIRT2 O/E cells to a greater degree than in the Previous studies have demonstrated that increased intracellular vector control cells. Similar results were also obtained in MCF7 ROS levels induce the nuclear localization and transcriptional cells following ectopic overexpression of SIRT2 (Supplementary activity of FOXO3A, resulting in upregulation of one of its Fig. S4A). This was associated with more induction of BIM levels targets, the proapoptotic, BH3 domain-only BIM protein (10, and increased caspase-3 cleavage and activity (Supplementary Fig. 11). Consistent with this, as compared with the untreated control S4B). In a dose- and time-dependent manner, treatment with cells, MB-231 cells overexpressing SIRT2 demonstrated higher H2O2 also induced markedly higher levels of cell death in MB-231 accumulation of FOXO3A in the nucleus (Fig. 6A and B). Treat- cells overexpressing SIRT2, associated with deacetylated and

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A MDA-MB-231 MDA-MB-231 B Vector SIRT2 O/E Vector SIRT2 O/E 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 0 0.5 2 4 h, H O 1.7 KD 2 2 MDA-MB-231 Hcells, O , 30 min 1048 2 2 1.6

1.5 720 1.4

1.3 480 1.2 MB-231/Vect 0 µmol/L MB-231/Vect 500 µmol/L 1.1 µ 242 Absorbance (320 nm) MB-231/SIRT2 0 mol/L 146 MB-231/SIRT2 500 µmol/L 1 IB: Anti-Prdx-1 IB: Anti-Prdx-1-SO3 0 20 40 60 β-Actin Time (min) D MCF7 MCF7 C 1.8 Vector SIRT2 O/E Vector SIRT2 O/E MCF7 Hcells, 2 O2 for min30 KD 0 50 100 200 0 50 100 200 0 50 100 200 0 50 100 200 μmol/L H22 O 1.7 1236 1048 1.6 1.5

720 1.4

1.3 MCF7/Vect 0 µmol/L 480 MCF7/Vect 200 µmol/L Absorbance (320 nm) 1.2 MCF7/SIRT2 0 µmol/L µ 242 MCF7/SIRT2 200 mol/L 146 1.1 0 12010080604020 IB: Anti-Prdx1 IB: Anti-Prdx1-SO3 β-Actin Time (min)

Figure 4.

SIRT2 overexpression increases the Prdx-1 chaperone activity in breast cancer cells following treatment with H2O2. A, MDA-MB-231 vector or SIRT2 O/E cells were treated with 500 mmol/L of H2O2 for 0–4 hours. At the end of treatment, the cells were lysed with native lysis buffer and separated on NuPAGE 3% to 8% tris-acetate native gels to detect oligomers and multimers of Prdx-1 (A, left) and oxidized Prdx-1 (A, right). Cell lysates were also probed with anti-b-actin to conﬁrm equal loading. B, MDA-MB-231 vector or SIRT2 O/E cells were treated with 500 mmol/L of H2O2 for 30 minutes. Then, cell lysates (500 mg) were collected and Prdx-1 was immunoprecipitated by anti-Prdx-1 (LF-MA0214; AbFrontier) and Dynabeads M-280 sheep anti-mouse IgG (Invitrogen). The immunoprecipitated Prdx-1 and Dynabeads were used for the chaperone activity assay. Each reaction contained 2 mmol/L MDH and the immunoprecipitated Prdx-1 with beads in 200 mLof 50 mmol/L HEPES-KOH buffer (pH 7.5). The absorbance at 320 nm was monitored utilizing a BioTek SynergyMx plate reader at 43C for 2 hours. C, MCF7 vector and

SIRT2 O/E cells were treated with the indicated concentrations of H2O2 for 30 minutes. At the end of treatment, the cells were lysed with native lysis buffer and separated on NuPAGE 3% to 8% tris-acetate native gels to detect oligomers and multimers of Prdx-1 (C, left) and oxidized Prdx-1 (C, right). Cell lysates were also

probed with anti-b-actin to conﬁrm equal loading. D, MCF7 vector and SIRT2 O/E cells were treated with the indicated concentrations of H2O2 for 30 minutes. Cell lysates were collected as in B and absorbance at 320 nm was monitored utilizing a BioTek SynergyMx plate reader at 43C for 2 hours, as above.

relatively inactive Prdx-1, as compared with a similar treatment culation failure, looping failure, and dorsal curvature (29). Next, with H2O2 of MB-231 cells expressing catalytically inactive we determined the effects of antisense morpholinos against exon mutant SIRT2 or those with KD of SIRT2 (Fig. 6C). Next, we 6 of the zebrafish homolog of SIRT2 or mismatch controls, compared the lethal effects of exposure to the intracellular ROS- injected into single-cell stage zebrafish embryos (29). The effects inducing agents, such as AT and menadione, in MB-231 and MCF- of the morpholinos on mRNA level of SIRT2 and the effect of 7 cells with the ectopic overexpression of SIRT2 versus the control treatment with 3.0 mmol/L of H2O2 for 30 minutes on ROS levels MB-231 and MCF7 cells with the ectopic expression of the vector at 48 hours postfertilization (hpf) were determined (Supplemen- alone. As shown in Fig. 6D and E, exposure to AT or menadione tary Fig. S5). Figure 7A demonstrates that compared with the induced significantly more lethality in cells with overexpression of mismatch control morpholino, antisense SIRT2 morpholino SIRT2 versus the control MB-231 and MCF7 cells. downmodulated mRNA levels of SIRT2. Treatment with H2O2 induced ROS levels in control embryos exposed to 3.0 mmol/L KD of SIRT2 reduces H2O2-mediated embryonic toxicity and H2O2, which was markedly suppressed by cotreatment with the cardiac abnormalities in zebrafish embryos antioxidant N-acetylcysteine (NAC; Fig. 7B). In contrast, SIRT2 Previous reports have documented that embryonic toxicity due morpholino treatment significantly attenuated ROS levels due to to oxidative stress induced by H2O2 or AT treatment of developing H2O2 treatment, which was further reduced by cotreatment with embryos of zebrafish (Danio rerio) is characterized by in vivo NAC (Fig. 7B). Notably, injection of the mismatch control mor- developmental abnormalities, including pericardial edema, cir- pholino, followed by exposure to 3.0 mmol/L H2O2, showed the

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MDA-MB-231 B A gH2AX DAPI MERGE MDA-MB-231 Vector SIRT2 O/E Vector m 2 2 Control 0 100 500 0 100 500 mol/L, H O , 2 Hours gH2AX SIRT2 O/E β-Actin

Vector

500 μmol/L H2O2 Tail movement 100 D Vector SIRT2 O/E 80 SIRT2 O/E SIRT2 Mutant 60 SIRT2 KD Control 40 H2O 2 (mmol/L), 24 Hours % of Cells of % C 0 200 400 20 0 MDA-MB-231 0–5 5–10 10–20 20–30 30–50 >50 Vector 100 Vector 80 SIRT2 O/E SIRT2 Mutant 200 mmol/L, H2O2 60 * SIRT2 KD 24 Hours MDA-MB-231 40 * SIRT2 O/E Cells of % * 20 * 0 0–5 5–10 10–20 20–30 30–50 >50 MDA-MB-231 100 Vector SIRT2 mutant 80 SIRT2 O/E SIRT2 Mutant m 2 2 400 mol/L, H O 60 SIRT2 KD 24 Hours * * 40 ** * MDA-MB-231 * *

% of Cells of % *** SIRT2- KD 20 ** * 0 0–5 5–10 10–20 20–30 30–50 >50

Figure 5. SIRT2 increases DNA damage on H2O2 exposure. A, MDA-MB-231 vector or SIRT2 O/E cells were plated in chamber slides overnight at 37 C. The following day, cells were treated with or without 500 mmol/L of H2O2 for 4 hours. After treatment, the cells were ﬁxed, permeabilized, and blocked with 3% BSA. The expression of gH2AX was detected by immunoﬂuorescent staining (red). Nuclei were stained with DAPI. B, MDA-MB-231 vector or SIRT2 O/E cells were treated with the

indicated concentrations of H2O2 for 2 hours. Following this, total cell lysates were immunoblotted for gH2AX and b-actin. C, MDA-MB-231 cells overexpressing SIRT2, SIRT2 (H187A) mutant, or SIRT2 KD were treated with the indicated concentration of H2O2 for 24 hours and comet assay was performed. D, quantitative tail movement of each indicated concentration in the MDA-MB-231 cells is presented. , values signiﬁcantly greater in cells treated with H2O2 than untreated control cells (P < 0.05).

characteristic toxicity profile, including pericardial edema, circu- ing the oncoproteins survivin, c-Myc, and cyclin D1 (33). SIRT2 is lation failure, and dorsal curvature in zebrafish larvae at 120 hpf. located at 19q13.2, which is frequently deleted in human gliomas However, consistent with the attenuation of ROS levels, SIRT2 (34). Consistent with these observations, SIRT2 knockout mice morpholino-treated embryos failed to exhibit developmental develop cancers, and SIRT2 levels are reduced in variety of cancer abnormalities including pericardial edema and dorsal curvature types, including breast, liver, renal, and prostate cancers (1, 5). (Fig. 7C). These findings demonstrate that even a partial KD of Along with SIRT2, the peroxide scavenger 2-Cys Prdx-1 has also SIRT2, by inducing acetylation and increased antioxidant activity been shown to have a tumor suppressive role and is classified as a of Prdx-1, abrogated the ROS-mediated toxic effects in zebrafish "gerontogene" (35). Mice lacking Prdx-1 develop tumors prema- embryos. turely during aging, associated with increased 8-oxo-dG levels and oxidative DNA damage (36). Prdx-1 deficiency was also shown to cause increased c-Myc and AKT activity, the latter due to ROS- Discussion mediated inactivation of PTEN (37–39). In addition, Prdx-1 was Previous reports have identified the role of SIRT2 as a tumor also demonstrated to inhibit TNFa-mediated NF-kB transcrip- suppressor, primarily due to its ability to maintain genomic tional activity (16). In the current study, we have established for fidelity during mitosis (1, 5, 6). SIRT2 achieves this by deacetylat- the first time a direct link between SIRT2 levels and Prdx-1 ing and stabilizing BUBR1 and APC/C activity, which delays deacetylation and activity. Utilizing DIGE coupled with MS/MS, anaphase until chromosomes are attached at the mitotic spindle we demonstrate that the predominantly cytosolic SIRT2 binds and (1, 5, 6, 32). Recently, SIRT2 was also shown to directly interact deacetylates cytosolic Prdx-1. This is consistent with the previous with b-catenin and inhibit the WNT transcriptional targets includ- reports, indicating that Prdx-1 present in the intermitochondrial

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SIRT2 Regulates Sensitivity to ROS in Breast Cancer

MDA-MB-231 A C FOXO3a DAPI MERGE 100 MDA-MB-231 100 Vector 80 Vector MDA-MB-231 Vector 80 SIRT2 O/E 60 SIRT2 O/E SIRT2 Mutant SIRT2 Mutant 60 40 SIRT2 KD 40 20 20 % PI-positive cells % PI-positive cells 0 0 Vector 02481624 + 500 μmol/L 0100250500 H O (mmol/L), Hours H O 2 2 2 2 H2O2 (mmol/L), 48 Hours D 100 100 MDA-MB-231 Vector MCF7 Vector P = 0.003 SIRT2 O/E 80 P < 0.05 80 MDA-MB-231 SIRT2 O/E MCF7 SIRT2 O/E 60 60 P = 0.01 40 40 20 20

SIRT2 O/E % PI-positive cells + 500 μmol/L 0 % PI-positive cells 0 013510 H2O2 013510 Arsenic trioxide (mmol/L), 48 hours Arsenic trioxide (mmol/L), 48 hours B Vector E 100 P MDA-MB-231 Vector = 0.004 MCF7 Vector Vector ** 100 80 P + 500 μmol/L *** 80 MDA-MB-231 SIRT2 O/E MCF7 SIRT2 O/E = 0.005 H O P = 0.003 60 2 2 60 SIRT2 O/E **** 40 40 SIRT2 O/E * 20 20 % PI-positive cells + 500 μmol/L % PI-positive cells 0 H O 0 2 2 0151015 051015 5 10 15 20 25 Menadione (mmol/L), 48 hours Menadione (mmol/L), 48 hours Mean fluorescence intensity

Figure 6.

SIRT2 overexpression induces cell death in breast cancer cells. A, MDA-MB231 vector and SIRT2 O/E cells were exposed to the indicated concentration of H2O2 for 4 hours, fixed, permeabilized, and stained for FOXO3a. Nuclei were stained with DAPI. Confocal immunofluorescent microscopy was performed using LSM 510Meta microscope (Zeiss) using a 63/1.2 NA oil immersion lens. B, quantification of mean fluorescent intensity of nuclear FOXO3A in MDA-MB-231 vector and SIRT2 O/E cells with or without treatment with H2O2 for 4 hours. , values significantly different in SIRT2-overexpressing cells with or without H2O2 treatment. , P < 0.05; , P < 0.005; , P < 0.0005; , P < 0.0001. C, MDA-MB-231 cells overexpressing SIRT2 were treated with indicated concentrations of H2O2 for 48 hours (left) or with 500 mmol/L of H2O2 for indicated times (right). The percentages of PI-positive, nonviable cells were determined by flow cytometry. D, MDA-MB-231 and MCF7 cells overexpressing SIRT2 and vector control were treated with the indicated concentrations of AT for 48 hours. At the end of treatment, the percentages of PI-positive, nonviable cells were determined by flow cytometry. E, MDA-MB-231 and MCF7 vector and SIRT2 O/E cells were treated with menadione as indicated for 48 hours. Following this, the percentage of PI-positive, nonviable cells in each condition was determined by flow cytometry.

space is deacetylated on lysine-197 by the mitochondria-resident the ultimate loss of viability caused by exposure of the SIRT2- SIRT3, whereas the cytosolic Prdx-1 is also a substrate for deace- overexpressing cells to high levels of H2O2 was due to increased tylation by HDAC6 (40, 41). Importantly, we demonstrate here nuclear accumulation of FOXO3A accompanied with the that SIRT2-mediated deacetylation of Prdx-1 reduces its antiox- induction of BIM levels (Fig. 7D). This is also consistent with idant peroxidase activity (Fig. 7D). Consequently, SIRT2-over- the tumor suppressor function of SIRT2. expressing breast cancer cells, especially when subjected to oxi- Compared with their normal counterparts, cancer cells dant stress induced by H2O2, accumulate ROS and DNA damage, exhibit increased levels of ROS, including superoxide and as estimated by the comet assay and increased gH2AX levels, as hydroxyl radicals and H2O2, which promotes cell signaling for well as demonstrate loss of cell viability. In contrast, this was proliferation and other biologic functions (13, 16, 42, 43). not seen in breast cancer cells with ectopic expression of the Engagement by the ligands or cytokines of receptor tyrosine catalytically inactive mutant form of SIRT2. Reduced peroxi- kinases or G protein–coupled receptors leads to transient dase activity of the deacetylated Prdx-1 was also associated generation of H2O2, catalyzed by the cell membrane-localized with ROS-induced overoxidation and multimer formation by NADPH oxidases (16, 42, 43). This H2O2 oxidizes and inacti- Prdx-1, which represents a switch from the peroxidase to vates cysteine residue in the nearby tyrosine phosphatases, chaperone function of Prdx-1 (15, 18, 27). Previous studies which normally attenuate receptor signaling by dephosphor- have shown that the chaperone function of multimeric Prdx-1 ylating the pathway signaling kinases, thereby promoting the enhances resistance to the lethal effects of oxidative stress and signaling for growth and proliferation (16, 17, 42, 43). Recent- heat shock (18, 27). Despite this, our ﬁndings also show that ly, membrane-associated Prdx-1 was shown to be transiently

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A 1.20 B 8,000 SIRT2 P P < 0.05 7,000 < 0.05 1.00 6,000 0.80 5,000 0.60 4,000 RFU 3,000 0.40 2,000 0.20 1,000

Relative mRNA expression 0.00 0 Figure 7. Control Control SIRT2 SIRT2 KD of SIRT2 in the zebrafish embryos MO MO+NAC MO MO+NAC Mismatch decreased H2O2-induced ROS levels and abrogated ROS-mediated cardiac +3 mmol/LH2O2 edema and abnormal body curvature. Uninjected A, the splice-blocking MO targeting Control MO SIRT2 -MO C Control against exon 6 (coding for the small domain of SIRT2) of zebrafish SIRT2 was injected into single-cell stage zebrafish embryos, and expression of Control SIRT2 mRNA was assessed from the embryos, after 48 hours, by qPCR. B, control and MO-treated embryos

were exposed to 3 mmol/L of H2O2 at 48 hpf with or without NAC. ROS levels in the embryos were monitored at 30 minutes using DCF-DA. C, the morphologic changes at day 5 (after +3 mmol/L H2O2 H2O2 treatment, 168 hpf) were imaged. D, schematic model for the activity of increased SIRT2 in breast cancer cells. Induction of SIRT2 decreases the Cell membrane antioxidant activity of Prdx-1, leading to D oxidation of Prdx-1 and increased ROS. This induces the transient induction of Prdx-1 multimers and increased Prdx-1 ROS-Inducing agents (e.g. AsO3) chaperone activity. The ROS that are Nucleus generated induce the translocation of FOXO3A into the nucleus, where it can SIRT2 Prdx-1 ROS Foxo3A activate the transcription of Antioxidant proapoptotic BIM. The ROS can also directly induce DNA damage, leading to Activity BIM increased cell death. Oxidized Foxo3A Prdx-1 Prdx-1 multimers Cell death ROS Prdx-1 DNA Chaperone Damage Activity

phosphorylated on its tyrosine-194 residue and thereby inacti- In contrast to its tumor suppressive role during tumorigenesis, vated, allowing nearby accumulation of H2O2,inactivationof in a variety of established tumor types, Prdx-1 levels are increased tyrosine phosphatases, and stimulation of the kinase-mediated and have been shown to be transcriptionally upregulated by NRF2 signaling (42). However, excessive levels of ROS can inﬂict (36, 38, 44). Increased Prdx-1 levels have been demonstrated in oxidative damage to lipids, proteins, and DNA (13). Among the many cancers, including bladder cancer and non–small cell lung H2O2 neutralizing proteins is Prdx-1, possessing a conserved N- cancer (NSCLC), where they have been associated with a high terminal cysteine residue, which is also oxidized by H2O2 but grade and advanced stages (35, 45, 46). In addition, Prdx-1 has reduced by thioredoxin (16, 43). Findings presented here been demonstrated to potentially serve as a prognostic and clearly demonstrate that KD of SIRT2, by inducing acetylation therapeutic target in cancer (47, 48). Overall, these reports high- of Prdx-1, increases its antioxidant peroxidase activity. This was light that Prdx-1 levels and activity regulate the redox homeosta- associated with a reduction in the DNA damage and apoptosis sis, which controls the growth and survival of cancer cells (13). triggered by H2O2-induced oxidant stress. Findings presented here clearly demonstrate that KD of SIRT2, by

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SIRT2 Regulates Sensitivity to ROS in Breast Cancer

inducing acetylation of Prdx-1, increases its antioxidant peroxi- Roche, Epizyme, and Boehringer. No potential conflicts of interest were dis- dase activity. This was accompanied by decreased in vitro accu- closed by the other authors. mulation of DNA damage detected by the comet assay, following exposure to oxidative stress induced either by H2O2 or by expo- Authors' Contributions sure to AT or menadione. Furthermore, in vivo SIRT2 KD also Conception and design: K.N. Bhalla exerted protection against toxicity associated with oxidative stress Development of methodology: H. Ma in zebrafish embryos. There was a reduction in the H O -induced Acquisition of data (provided animals, acquired and managed patients, 2 2 provided facilities, etc.): W. Fiskus, V. Coothankandaswamy, J. Chen, K. Ha, accumulation of ROS and the embryos also failed to develop D.T. Saenz, S.S. Krieger, C.P. Mill, B. Sun, P. Huang characteristic features of embryonic toxicity due to oxidative Analysis and interpretation of data (e.g., statistical analysis, biostatistics, stress. computational analysis): W. Fiskus, V. Coothankandaswamy, J. Chen, H. Ma, In summary, our findings demonstrate that in cancer cells, D.T. Saenz, S.S. Krieger, B. Sun, J.S. Mumm, K.N. Bhalla selectively activating SIRT2 would lead to deacetylation and Writing, review, and/or revision of the manuscript: W. Fiskus, P. Huang, inactivation of Prdx-1, thereby sensitizing cancer cells to agents J.S. Mumm, A.M. Melnick, K.N. Bhalla Study supervision: K.N. Bhalla that induce oxidative stress and promote lethal DNA damage. By inducing nuclear accumulation of FOXO3A and induction of BIM, increased SIRT2 with reduced Prdx-1 activities could also Grant Support induce lethal effects through a mechanism independent of the This research was partially supported by the CCSG P30 CA016672. The costs of publication of this article were defrayed in part by the payment function of the tumor suppressor TP53. of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conflicts of Interest A. Melnick reports receiving a commercial research grant from Janssen, Received January 13, 2016; revised May 29, 2016; accepted June 21, 2016; Roche, and Eli Lilly and is a consultant/advisory board member for Eli Lilly, published OnlineFirst August 8, 2016.

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SIRT2 Deacetylates and Inhibits the Peroxidase Activity of Peroxiredoxin-1 to Sensitize Breast Cancer Cells to Oxidant Stress-Inducing Agents

Warren Fiskus, Veena Coothankandaswamy, Jianguang Chen, et al.

Cancer Res 2016;76:5467-5478. Published OnlineFirst August 8, 2016.

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